Abundance, Distribution, and Activity of Fe(II)-Oxidizing and Fe(III)-Reducing Microorganisms in Hypersaline Sediments of Lake Kasin, Southern Russia

The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter-1) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26×102 to 8.32×103 iron(II)-oxidizing Bacteria and 4.16×102 to 2.13×103 iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria, total Archaea, and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense, Desulfosporosinus lacus, and members of the Bacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl

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

NaCl and KCl mediate log increase in AAV vector particles and infectious titers in a specific/timely manner with the HSV platform

The increasing demand for adeno-associated virus (AAV) vectors, a result from the surging interest for their potential to cure human genetic diseases by gene transfer, tumbled on low-performing production systems. Innovative improvements to increase both yield and quality of the vector produced have become a priority undertaking in the field. In a previous study, we showed that adding a specific concentration of sodium chloride (NaCl) to the production medium resulted in a dramatic increase of AAV vector particle and infectious titers when using the herpes simplex virus (HSV) production system, both in adherent or suspension platforms. In this work, we studied additional salts and their impact on AAV vector production. We found that potassium chloride (KCl), or a combination of KCl and NaCl, resulted in the highest increase in AAV vector production. We determined that the salt-mediated effect was the most impactful when the salt was present between 8 and approximately 16 h post-infection, with the highest rate increase occurring within the first 24 h of the production cycle. We showed that the AAV vector yield increase did not result from an increase in cell growth, size, or viability. Furthermore, we demonstrated that the impact on AAV vector production was specifically mediated by NaCl and KCl independently of their impact on the osmolality of the production media. Our findings convincingly showed that NaCl and KCl were uniquely efficacious to promote up to a 10-fold increase in the production of highly infectious AAV vectors when produced in the presence of HSV. We think that this study will provide unique and important new insights in AAV biology toward the establishment of more successful production protocols

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