APOBEC3-mediated hypermutation of retroviral vectors produced from some retrovirus packaging cell lines.

APOBEC3 proteins are packaged into retrovirus virions and can hypermutate retroviruses during reverse transcription. We found that HT-1080 human fibrosarcoma cells hypermutate retroviruses, and that the HT-1080 cell-derived FLYA13 retrovirus packaging cells also hypermutate a retrovirus vector produced using these cells. We found no hypermutation of the same vector produced by the mouse cell-derived packaging line PT67 or by human 293 cells transfected with the vector and retrovirus packaging plasmids. We expect that avoidance of vector hypermutation will be particularly important for vectors used in gene therapy, wherein mutant proteins might stimulate deleterious immune responses.

Capsid-expressing DNA in AAV vectors and its elimination by use of an oversize capsid gene for vector production.

Adeno-associated virus (AAV) vectors have been shown to mediate persistent transduction in animal models of gene therapy. However, clinical trials with AAV vectors have shown that an immune response to AAV capsid protein can result in clearance of transduced cells. One source of capsid antigen is from the delivered vector virions, but expression of cap DNA impurities in AAV vector preparations might provide an alternative and persistent source of capsid antigen. Here we show that DNA without any AAV sequences can be packaged in AAV virions, and that both cap and rep DNA are packaged into AAV vectors produced by standard methods. Using a sensitive complementation assay, we also observed significant expression of capsid in cultured cells transduced with AAV vectors. In an attempt to solve this problem, we inserted a large intron into the cap gene to generate a capsid expression cassette (captron) that is too large for packaging into AAV virions. Both complementation assays and quantitative reverse-transcription PCR analysis showed that cultured cells infected with AAV vectors made with the captron plasmid expressed no detectable capsid. Elimination of transfer of capsid-expressing DNA may reduce immune responses to AAV vector-transduced cells and promote long-term expression of therapeutic proteins.

An assessment of long term ecosystem research activities across European socio-ecological gradients.

Integration of European long term ecosystem research (LTER) would provide important support for the management of the pan-European environment and ecosystems, as well as international policy commitments. This does require appropriate coverage of Europe and standardised frameworks and research methods between countries. Emerging interest in socio-ecological systems prompted the present assessment of the distribution of LTER activities across European socio-ecological gradients. This paper presents a European stratification with a 1 km(2) resolution, delineating 48 broad socio-ecological regions. The dataset is based on an existing biogeophysical stratification constructed using multivariate clustering of mainly climatic variables and a newly developed socio-economic stratification based on an economic density indicator. The coverage of European LTER facilities across the socio-ecological gradients is tested using this dataset. The analysis shows two strong biases in the present LTER effort. Firstly, urban and disturbed regions are consistently under-represented, illustrating a bias for traditional ecological research away from human activity. Secondly, the Mediterranean, for which some of the most extreme global change impacts are projected, is receiving comparatively little attention. Both findings can help guide future investment in the European LTER network - and especially in a Long Term Socio-Ecological Research (LTSER) component- to provide a more balanced coverage. This will provide better scientific understanding of pan-European environmental concerns and support the management of natural resources and international policy commitments in the European Union.

Talar neck fractures and rates of avascular necrosis.

Talar neck fractures are unique and potentially debilitating injuries. Their successful treatment requires an understanding of talar anatomy and arterial blood supply, as well as knowledge of the sequelae of these injuries, particularly avascular necrosis of the talar body. Presented is an extensive literature review on talar neck fractures and Hawkins' classification, with special emphasis on the rates of avascular necrosis as determined by selected researchers on the topic.

Randomized, controlled trial of diabetic patient education: improved knowledge without improved metabolic status.

We randomized 749 insulin-treated patients on the rolls of the Mount Sinai Medical Center Diabetes Clinic in a controlled trial of diabetic patient education; 345 agreed to participate, of whom 165 were assigned to the education group and 180 to the control group. Cognitive scores increased from 5.3 +/- 1.6 to 5.8 +/- 1.6 in the education group, but there was no change in the control group, whose score was 5.3 +/- 1.7 before and after the intervention (P = .0073). HbA1c fell from 6.8 +/- 2.1 to 6.1 +/- 2.0% in the education group and from 6.6 +/- 2.0 to 6.3 +/- 2.0% in the control group, an insignificant difference (P = .1995). The fasting blood glucose decreased from 223 +/- 94 to 179 +/- 73 mg/dl in the education group and from 199 +/- 81 to 185 +/- 76 mg/dl in the controls (P = .1983). Triglycerides, high- and low-density lipoprotein cholesterol, and insulin dosage also failed to show significant variation among groups. The foot-lesion score showed similar progression in the education and control groups. Neither diastolic nor systolic blood pressure showed significantly greater change in the education or the control group, with falls noted, particularly in diastolic pressures, in both patient groups. Differences between the groups were not significant for sick days, hospitalizations, emergency room visits, or outpatient visits. The sample sizes of the study and control populations were sufficiently large to detect a difference in means between the education and control groups in the HbA1c, the primary outcome variable, of greater than 1.0%, with alpha = .05 and a power of .95. Thus, our study suggests that patient education may not be an efficacious therapeutic intervention in most adults with insulin-treated diabetes mellitus.