Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization

Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography

Safety and Immunogenicity of an HIV-1 Gag DNA Vaccine with or without IL-12 and/or IL-15 Plasmid Cytokine Adjuvant in Healthy, HIV-1 Uninfected Adults

DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.Clinicaltrials.gov NCT00115960 NCT00111605

A study of the distobuccal root canal orifice of the maxillary second molars in Chinese individuals evaluated by cone-beam computed tomography

As is commonly understood, the root canal morphology of the maxillary molars is usually complex and variable. It is sometimes difficult to detect the distobuccal root canal orifice of a maxillary second molar with root canal treatment. No literature related to the distobuccal root canals of the maxillary second molars has been published. Objective: To investigate the position of the distobuccal root canal orifice of the maxillary second molars in a Chinese population using cone-beam computed tomography (CBCT). Material and methods: In total, 816 maxillary second molars from 408 patients were selected from a Chinese population and scanned using CBCT. The following information was recorded: (1) the number of root canals per tooth, (2) the distance between the mesiobuccal and distobuccal root canal orifice (DM), (3) the distance between the palatal and distobuccal root canal orifice (DP), (4) the angle formed by the mesiobuccal, distobuccal and palatal root canal orifices (∠PDM). DM, DP and ∠PDM of the teeth with three or four root canals were analyzed and evaluated. Results: In total, 763 (93.51%) of 816 maxillary second molars had three or four root canals. The distance between the mesiobuccal and distobuccal orifice was 0.7 to 4.8 mm. 621 (81.39%) of 763 teeth were distributed within 1.5-3.0 mm. The distance between the palatal and distobuccal orifice ranged from 0.8 mm to 6.7 mm; 585 (76.67%) and were distributed within 3.0-5.0 mm. The angle (∠PDM) ranged from 69. 4º to 174.7º in 708 samples (92.80%), the angle ranged from 90º to 140º. Conclusions: The position of the distobuccal root canal orifice of the maxillary second molars with 3 or 4 root canals in a Chinese population was complex and variable. Clinicians should have a thorough knowledge of the anatomy of the maxillary second molars