Lesion-dependent regulation of transgene expression in the rat brain using a human glial fibrillary acidic protein-lentiviral vector.

The ability to regulate transgene expression will be crucial for development of gene therapy to the brain. The most commonly used systems are based on a transactivator in combination with a drug, e.g. the tetracycline-regulated system. Here we describe a different method of transgene regulation by the use of the human glial fibrillary acidic protein (GFAP) promoter. We constructed a lentiviral vector that directs transgene expression to astrocytes. Using toxin-induced lesions we investigated to what extent transgene expression could be regulated in accordance with the activation of the endogenous GFAP gene. In animals receiving excitotoxic lesions of the striatum we detected an eightfold increase of green fluorescent protein (GFP)-expressing cells. The vast majority of these cells did not divide, suggesting that the transgene was indeed regulated in a similar fashion as the endogenous GFAP gene. This finding will lead to the development of lentiviral vectors with autoregulatory capacities that may be very useful for gene therapy to the brain

Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

<p>Abstract</p> <p>Background</p> <p>Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary elements that can prevent enhancer-promoter interactions, if placed between these elements, and protect transgene cassettes from silencing and positional effects. It has been suggested that insulators can improve the safety and performance of lentiviral vectors. Therefore insulators have been incorporated into lentiviral vectors in order to enhance their safety profile and improve transgene expression. Commonly such insulator vectors are produced at lower titers than control vectors thus limiting their potential use.</p> <p>Results</p> <p>In this study we cloned in tandem copies of the chicken β-globin insulator (cHS4) on both sides of the transgene cassette in order to enhance the insulating effect. Our insulator vectors were produced at significantly lower titers compared to control vectors, and we show that this reduction in titer is due to a block during the transduction process that appears after reverse transcription but before integration of the viral DNA. This non-integrated viral DNA could be detected by PCR and, importantly, prevented efficient transduction of target cells.</p> <p>Conclusion</p> <p>These results have importance for the future use of insulator sequences in lentiviral vectors and might limit the use of insulators in vectors for <it>in vivo </it>use. Therefore, a careful analysis of the optimal design must be performed before insulators are included into clinical lentiviral vectors.</p

A General Chemical Method to Regulate Protein Stability in the Mammalian Central Nervous System

SummaryThe ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system

Migration and risk of intellectual disability with and without autism:A population-based cohort study

Objective To investigate whether parental migration, parental region of origin, timing of child's birth in relation to maternal migration and parental reason for migration are associated with intellectual disability (ID) with and without autism. Methods We used a register-based cohort of all individuals aged 0–17 years in Stockholm County during 2001–2011. General estimating equation logistic model and additionally sibling comparison were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). The models were adjusted for child's sex and birth year and parental age at child's birth, and additionally for migrant-specific variables in the analyses including only children with migrant parent(s). Results Within the eligible sample of 670,098 individuals, 3781 (0.6%) had ID with autism, and 5076 (0.8%) had ID without autism. Compared with children with Swedish-born parents, children with both parents born abroad had an increased risk of ID with autism (OR = 1.6, CI 1.5–1.8) and ID without autism (OR = 1.9, CI 1.7–2.0). Among these children with both parents born abroad, it was protective of ID with autism when the child's birth occurred before and later than four years after maternal migration, which was replicated in the sibling comparison. The associations with both conditions were more pronounced with parental origin in regions comprising low- and middle-income countries and with reasons other than work or study. Conclusions Parental migration is associated with ID regardless of co-occurrence of autism. Our results indicate an association between environmental factors during pregnancy related to migration and offspring ID with autism, although further confirmative studies are needed

Maternal smoking and smokeless tobacco use during pregnancy and offspring development:Sibling analysis in an intergenerational Swedish cohort

BACKGROUND: The association between maternal smoking in pregnancy and offspring intellectual disability (ID) is less well understood than that of smoking and fetal growth restriction. As fetal growth and cognitive development may share similar confounding structures, comparison of the two associations may improve understanding of the causal nature of the association with ID. Furthermore, comparisons of smoking with smokeless tobacco use may aid identification of mechanisms of action. METHODS: This was a cohort study of all Swedish births between 1999 and 2012 (n = 1 070 013), with prospectively recorded data. We assessed the association between maternal smoking during pregnancy and offspring outcomes ID and born small for gestational age (SGA). Analyses were repeated for snus use in pregnancy. Using a sibling design, we estimated within-family effects that control for shared sibling characteristics. RESULTS: Those exposed to maternal smoking in pregnancy had increased odds of ID [odds ratio (OR) = 1.24, 95% confidence interval (CI): 1.16-1.33] and SGA (OR = 2.19, 95% CI: 2.11-2.27) after confounder adjustment. Within-family effects were found for SGA (OR = 1.44, 95% CI: 1.27-1.63) but not ID (OR = 0.92, 95% CI: 0.74-1.14). For snus use, the results for ID were similar to smoking. We found increased odds of offspring SGA among mothers who used snus in pregnancy in sensitivity analyses but not in primary analyses. CONCLUSIONS: Our findings are consistent with a causal effect of maternal smoking in pregnancy on risk of offspring born SGA but not on risk of ID. We found no evidence for a causal effect of snus use in pregnancy on ID and inconclusive evidence for SGA

Dangerous dads? Ecological and longitudinal analyses of paternity leave and risk for child injury

BACKGROUND: In 1974, Sweden became the first country to permit fathers to take paid parental leave. Other countries are currently following suit issuing similar laws. While this reform supports the principles of the United Nations convention of the right for children to be with both parents and enshrines the ethos of gender equality, there has been little systematic examination of its potential impact on child health. Instead, there is uninformed debate that fathers may expose their children to greater risks of injury than mothers. In this Swedish national study, the authors therefore assess whether fathers' parental leave can be regarded as a more serious risk factor for child injuries than that of mothers. METHODS: Nationwide register-based ecological and longitudinal studies of hospitalisation due to injury (and intoxication) in early childhood, involving the Swedish population in 1973-2009 (ecological design), and children born in 1988 and 1989 (n=118 278) (longitudinal design). RESULTS: An increase in fathers' share of parental leave over time was parallelled by a downward trend in child injury rates (age 0-4 years). At the individual level, the crude incidence of child injury (age 0-2 years) was lower during paternity as compared with maternity leave. This association was, however, explained by parental socio-demographic characteristics (multivariate HR 0.96, 95% CI 0.74 to 1.2). CONCLUSION: There is no support for the notion that paternity leave increases the risk of child injury.The Swedish Council for Working Life and Social Research and the Swedish Research Council.http://jech.bmj.com/hb2013ay201

Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500mL/min, with a growth rate of 0.74 +/- 0.13h(-1) which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8%+/- 2.4% vs. 11.5%+/- 4.3%), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs. 3.59 for N-2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5 ' nucleotidase activity (370U/L vs. 240U/L in N-2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimization of L. reuteri DSM 17938

Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500 ml/min, with a growth rate of 0.74 ± 0.13 h-1 which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8 ± 2.4 % vs 11.5 ± 4.3 %), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs 3.59 for N2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5’nucleotidase activity (370 U/L vs 240 U/L in N2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90 min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimisation of L. reuteri DSM 17938