8 research outputs found
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Construction and Development of a BF3 Neutron Detector At Brookhaven National Laboratory (BNL)
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Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material
A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus–free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 × 1011 particles/ml; 95% confidence interval [CI], 7.89 × 1011 to 1.05 × 1012 particles/ml), vector genomes ({X}, 3.28 × 1010 vector genomes/ml; 95% CI, 2.70 × 1010 to 4.75 × 1010 vector genomes/ml), transducing units ({X}, 5.09 × 108 transducing units/ml; 95% CI, 2.00 × 108 to 9.60 × 108 transducing units/ml), and infectious units ({X}, 4.37 × 109 TCID50 IU/ml; 95% CI, 2.06 × 109 to 9.26 × 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed
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Progressive Excitability Changes in the Medial Entorhinal Cortex in the 3xTg Mouse Model of Alzheimers Disease Pathology.
Alzheimers disease (AD) is a chronic neurodegenerative disorder characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in hippocampus, but less is known about changes in medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at 3 and 10 months of age in the 3xTg mouse model of AD pathology, using male and female mice. At 3 months of age, before the onset of memory impairments, we found early hyperexcitability in intrinsic properties of MECII stellate and pyramidal cells, but this was balanced by a relative reduction in synaptic excitation (E) compared with inhibition (I; E/I ratio), suggesting intact homeostatic mechanisms regulating MECII activity. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable, and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsic and synaptic hyperexcitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this postsymptomatic time point, which may contribute to the emergence of memory deficits in AD.SIGNIFICANCE STATEMENT AD causes cognitive deficits, but the specific neural circuits that are damaged to drive changes in memory remain unknown. Using a mouse model of AD pathology that expresses both amyloid and tau transgenes, we found that neurons in the MEC have altered excitability. Before the onset of memory impairments, neurons in layer 2 of MEC had increased intrinsic excitability, but this was balanced by reduced inputs onto the cell. However, after the onset of memory impairments, stellate cells in MEC became further hyperexcitable, with increased excitability exacerbated by increased synaptic inputs. Thus, it appears that MEC stellate cells are uniquely disrupted during the progression of memory deficits and may contribute to cognitive deficits in AD
The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time
SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation
Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material
Here, the AAV Reference Standard Working Group presents the results of their international collaboration to produce and characterize a recombinant AAV serotype 2 Reference Standard Material. The purpose of this material is to provide a universal standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors