Nanoscale Subsynaptic Domains Underlie the Organization of the Inhibitory Synapse

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Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus

Gene delivery vectors based on adeno-associated virus (AAV) have significant therapeutic potential, but much room for improvement remains in the areas of vector engineering and production. AAV production requires complementation with either helper virus, such as adenovirus, or plasmids containing helper genes, and helper virus-based approaches have distinct advantages in the use of bioreactors to produce large quantities of AAV vectors for clinical applications. However, helper viruses must eventually be inactivated and removed from AAV preparations to ensure safety. The current practice of thermally inactivating adenovirus is problematic as it can also inactivate AAV. Here, we report a novel method using high hydrostatic pressure (HHP) to selectively and completely inactivate helper adenovirus without any detectable loss of functional AAV vectors. The pressure inactivation kinetics of human adenovirus serotype 5 and the high-pressure stabilities of AAV serotypes 2 and 5 (AAV2, AAV5), which were previously unknown, were characterized. Adenovirus was inactivated beyond detection at 260 MPa or higher, whereas AAV2 was stable up to approximately 450 MPa, and surprisingly, AAV5 was stable up to at least 700 MPa. The viral genomic DNA of pressure-inactivated AAV2 was made sensitive to DNAse I digestion, suggesting that gross changes in particle structure had occurred, and this hypothesis was further supported by transmission electron microscopy. This approach should be useful in the laboratory- and clinical-scale production of AAV gene delivery vectors. Moreover, HHP provides a tool for probing the biophysical properties of AAV, which may facilitate understanding and improving the functions of this important virus.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Assessing enzyme activities using stable isotope labeling and mass spectrometry

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.