Transient expression of genes delivered to newborn rat liver using recombinant adeno‐associated virus 2/8 vectors

Background In vivo adeno‐associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver. Methods Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2‐day‐old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and β‐galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected β‐galactosidase clusters. Results In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, β‐galactosidase expression was maximum at day 7, but only a few (less than 1%) β‐galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75‐fold higher in positive cell clusters than in the surrounding parenchyma. Conclusions The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period. Copyright © 2009 John Wiley & Sons, Ltd.

Transit‐time method to demonstrate whether or not an impedance matching occurs at the pulmonary junction

Comparison of methods to assess quadriceps muscle volume using magnetic resonance imaging

Purpose To compare the precision of four methods to estimate the volume of quadriceps muscles using axial MRI. Materials and Methods Entire legs of 10 healthy young subjects were scanned using a 1.5 Tesla magnetic resonance imaging scanner and 4‐mm‐thick sections without any gaps. Quadriceps muscles were outlined on all of the slices to obtain the MRI reference standard measure of quadriceps muscle volume. This MRI reference standard was compared with the volume estimated using (i) the truncated cone formula, (ii) the Cavalieri method, (iii) a cubic spline interpolation of missing cross sectional areas, and, (iv) the deformation of a parametric specific object. For each method, 3 to 21 slices were used. Results The average volume error was significantly ( P < 0.001) different in comparing the four methods (4.4%, 2.3%, 1.1%, and 1.2%, respectively). In addition, the number of slices required to reach a given volume error was significantly ( P < 0.001) different across all methods (respectively, 12, 9, 5, and 7 slices required to reach a volume error of 1.1%). Conclusion While methods based on interpolation and deformation of a parametric specific object have not been used in literature, these two methods are the most precise approaches to reach a given level of precision. J. Magn. Reson. Imaging 2009;30:1116–1123. © 2009 Wiley‐Liss, Inc.

Online H/D exchange liquid chromatography as a support for the mass spectrometric identification of the oxidation products of melatonin

The hydrogen–deuterium exchange of protonated melatonin and its in vitro oxidation end‐products have been examined by liquid chromatography coupled with ion‐trap mass spectrometry. Specific H/D scrambling of protons in the C2 and C4 positions of the indole ring during gas‐phase fragmentation process was observed for both melatonin and its oxidation products. Collision‐induced dissociation spectra showed losses of variably deuterated NH 3 , H 2 O and CH 3 CONH 2 . In addition, a similar H/D scrambling behaviour was observed for the oxidation products, obtained from the opening of the indole ring by oxidative attack. Fragmentation pathways are proposed and H/D scrambling has been employed as a fingerprint, allowing identification of N 1 ‐acetyl‐5‐methoxykynurenin (AMK), N 1 ‐acetyl‐ N 2 ‐formyl‐5‐methoxykynurenin (AFMK), dehydro‐AFMK and hydroxymelatonin as the oxidation products of melatonin in vitro . Copyright © 2008 John Wiley & Sons, Ltd.

Influence of the HCV subtype on the virological response to pegylated interferon and ribavirin therapy

The hepatitis C virus genotype is considered to be the most important baseline predictor of a sustained virological response in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. The influence of the subtype on the sustained virological response was investigated in patients infected with genotypes 1, 4, 5, or 6. This study was done on 597 patients with chronic hepatitis C who were given pegylated interferon and ribavirin for 48 weeks. The overall rate of sustained virological response in the 597 patients was 37.8%. Univariate analysis indicated that the sustained virological response of patients infected with subtype 1b (39%) tended to be higher than that of patients infected with subtype 1a (30.6%; P  = 0.06) and it was similar to those patients infected with subtypes 4a (51.3%; P  = 0.12) or 4d (51.7%; P  = 0.16). Multivariate analysis indicated that five factors were independently associated with sustained virological response: the age (OR 0.97; 95% CI = 0.95–0.99), absence of cirrhosis (OR: 2.92; 95% CI = 1.7–5.0; P  < 0.01), absence of HIV co‐infection (OR: 2.08; 95% CI = 1.2–3.5; P  < 0.01), low baseline plasma HCV RNA concentration (OR: 1.74; 95% CI = 1.2–2.6; P  < 0.01), and the subtype 1b (OR: 1.61; 95% CI = 1.0–2.5; P  = 0.04) or subtypes 4a and 4d (OR: 2.03; 95% CI = 1.1–3.8; P  = 0.03). In conclusion, among difficult‐to‐treat genotypes, the subtype 1a is associated with a lower response to anti‐HCV therapy than subtypes 1b, 4a, and 4d. J. Med. Virol. 81:2029–2035, 2009. © 2009 Wiley‐Liss, Inc.

A deficit of brain dystrophin 71 impairs hypothalamic osmostat

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end‐feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71‐null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71‐null mice was lower than that of wild‐type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild‐type was not observed in Dp71‐null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71‐null than in wild‐type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end‐feet localization emphasizes the importance of neuronal–vascular–glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the “osmostat,” whereas, in the neurohypopohysis, it may be involved in fine‐tuning AVP release. © 2009 Wiley‐Liss, Inc.

The surface radiation budget over North America: gridded data assessment and evaluation of regional climate models

While surface station observations of downwelling radiation offer accuracy at high temporal resolution, they do not easily allow an evaluation of model surface radiation budgets (SRB) over a wide geographical area. We evaluate three gridded SRB data sets against detailed observations from six surface radiation sites from the US surface radiation (SURFRAD) network. We subsequently use the most accurate surrogate observational data set for evaluation of model‐simulated SRB. The data sets assessed are: ERA40—reanalysis of European Centre for Medium‐Range Weather Forecasts (ECMWF), North American Regional Reanalysis (NARR)—regional reanalysis of National Centres for Environmental Prediction (NCEP) and the surface radiative budget (SRB) from the International Satellite Cloud Climatology Project (ISCCP). Due to varying constraints with respect to temporal coverage of each data set, the evaluation period used in this study is 1996–2001, inclusive. The ERA40 downwelling longwave radiation (DLR) appears the most accurate surrogate observation, while both ERA40 and ISCCP show accurate results when the incoming shortwave radiation (ISR) is considered across the annual cycle. Winter DLR is less accurate in ISCCP with a positive bias and lack of very low (<200 Wm −2 ) flux values. The NARR SRB shows a large positive bias in the ISR throughout the annual cycle, linked to a significant underestimate of cloud cover. The ERA40 data are subsequently used to evaluate the simulated SRB in three regional climate models across North America. With respect to solar radiation, cloud cover biases are seen to be crucial, while for longwave fluxes both cloud fraction and in‐cloud water content are important to simulate correctly. Inclusion of trace gases beyond H 2 O, CO 2 and O 3 appears necessary for an accurate calculation of clear‐sky longwave radiation. Error compensation frequently occurs between the various components contributing to a model total‐sky SRB. This is important to consider when trying to identify the underlying causes of errors in the simulated total SRB. Copyright © 2009 Royal Meteorological Society

Optimization of a gastrointestinal model applicable to the evaluation of bioaccessibility in fish feeds

BACKGROUND: Although several types of in vitro digestibility assays have been applied to nutritional evaluation of feeds for aquatic organisms, all of them are based on the use of closed reactors and do not simulate the gastric phase of the digestion. Our objective was to evaluate the suitability of a gastrointestinal model based on the use of a digestion cell provided by a semi‐permeable reaction chamber, which allows continuous removal of digestion products as they are produced. We tested the effects of some factors, like the inclusion of a gastric phase, reaction temperature or bile salts on the hydrolysis of feed proteins by fish enzymes. RESULTS: We found that the most suitable operational conditions to simulate the digestion process must include a short acid pre‐digestion as well as the use of bile salts in the reaction mixture. Acid pre‐digestion resulted in a significant increase in the liberation of amino acids which represented more than twice that measured when using a single phase. The addition of two bile salts (45 µmol L −1 sodium taurocholate + chenodesoxycolate) resulted in almost a threefold increase in the hydrolysis of feed protein. The use of the described open system also allows the evaluation of carbohydrate hydrolysis as well as determination of residual undigested matter, in a similar manner to that carried out in ruminants with the DAISY system. CONCLUSION: Results suggest the system can be a very suitable model for evaluation of bioaccessibility in fish feeds. Copyright © 2009 Society of Chemical Industry

Effect of drying and storage on the degradation of total carotenoids in orange‐fleshed sweetpotato cultivars

BACKGROUND: Orange‐fleshed sweetpotato (OFSP) can be used to tackle vitamin A deficiency, a major public health problem in most developing countries. In East Africa, common ways of using sweetpotato include drying and subsequent storage. The aim of the study was to investigate the impact of drying and storage on the total carotenoid retention (as an estimate of provitamin A retention) from OFSP. RESULTS: Losses of total carotenoid during drying were generally low (15% or less). Total carotenoid retention in OFSP was not dependent on the type of dryer (solar or sun). Sweetpotato cultivar (Ejumula, Kakamega, SPK004/1, SPK004/1/1, SPK004/6 or SPK004/6/6) had a significant effect on retention in drying ( P < 0.05). High percentage losses of total carotenoids were, however, correlated with high moisture content and high carotenoid content in fresh sweetpotato roots. After 4 months' storage at room temperature in Uganda, losses of total carotenoid in dried sweetpotato chips were high (about 70%) and this was not dependent on the use of opaque or transparent packaging. CONCLUSION: Losses of carotenoids during storage were considered to be more of a nutritional constraint to the utilisation of dried sweetpotato than losses occurring during drying. The relationship between characteristics of the cultivars and losses of carotenoids during drying should be taken into account in selection of cultivars for processing. Copyright © 2009 Society of Chemical Industry

Antiangiogenic agents and late anastomotic complications

Anticancer agents targeting circulating vascular endothelial growth factor (VEGF) (e.g., bevacizumab and aflibercept) are strong angiogenesis inhibitors. As such, they may hamper the healing process, notably in the early postoperative period. Whether antiangiogenic agents may be associated with late postoperative healing complications is less known. We reviewed three cases of patients with anastomotic complications under antiangiogenic treatment occurring more than 1 year after initial surgery and we conducted a review of the literature. We report the first case of delayed anastomotic leakage which occurred under aflibercept therapy 13 months after a bilioenteric anastomosis and two cases of delayed rectal anastomotic complications associated with bevacizumab treatment 18 and 78 months after surgery. Fifteen similar cases of late gastrointestinal anastomotic complications were found in the English literature. Antiangiogenic agents are probably not deleterious to a healed wound. However, they appear to be associated with an increased risk of complications in a subgroup of patients. According to the 18 cases reported, the main risk factors appear to be low anterior resection for rectal cancer, perioperative radiotherapy, and early postoperative leak which heals through the formation of abundant and hypervascularized granulation tissue. J. Surg. Oncol. 2010;101:180–183. © 2009 Wiley‐Liss, Inc.