Validation and Retrospective Clinical Evaluation of a Quantitative 16S rRNA Gene Metagenomic Sequencing Assay for Bacterial Pathogen Detection in Body Fluids.

Next-generation sequencing-based 16S rRNA gene metagenomic sequencing (16S MG) technology has tremendous potential for improving diagnosis of bacterial infections given its quantitative capability and culture-independent approach. We validated and used a quantitative 16S MG assay to identify and quantify bacterial species in clinical samples from a wide spectrum of infections, including meningitis, septic arthritis, brain abscess, intra-abdominal abscess, soft tissue abscess, and pneumonia. Twenty clinical samples were tested, and 16S MG identified a total of 34 species, compared with 22 species and three descriptive findings identified by culture. 16S MG results matched culture results in 75% (15/20) of the samples but detected at least one more species in five samples, including one culture-negative cerebrospinal fluid sample that was found to contain Streptococcus intermedius. Shotgun metagenomic sequencing verified the presence of all additional species. The 16S MG assay is highly sensitive, with a limit of detection of 10 to 100 colony-forming units/mL. Other performance characteristics, including linearity, precision, and specificity, all met the requirements for a clinical test. This assay showed the advantages of accurate identification and quantification of bacteria in culture-negative and polymicrobial infections for which conventional microbiology methods are limited. It also showed promises to serve unmet clinical needs for solving difficult infectious diseases cases.

Topography of bovine papillomavirus E2 protein on the viral genome during the cell cycle.

The multifunctional papillomavirus E2 protein serves important roles in transcriptional activation and genome maintenance and cooperates with the viral E1 helicase for the initiation of viral DNA replication. The bovine papillomavirus genome contains seventeen E2 binding sites, largely concentrated within the long control region, and a single E1 binding site at the origin of viral replication. Using chromatin immunoprecipitation (ChIP) followed by restriction enzyme digestion and PCR, we show that BPV E1 was present only in the region of an active origin of replication and that BPV E2 remained attached to definable segments of the viral genome at specific stages of the cell cycle.

Tumor necrosis factor alpha leads to increased cell surface expression of CXCR4 in SK-N-MC cells.

Both host and viral factors play an important role in the pathogenesis of human immunodeficiency virus (HIV)-associated bran injury. In this study, the authors examined the interactions between tumor necrosis factor (TNF)-alpha, CXCR4, the alpha chemokine receptor, and three HIV isolates, including the T-tropic viruses, HIV-1(MN) and HIV-1(IIIB), and the dual tropic virus, HIV-1(89.6). The authors show by flow cytometry that treatment of differentiated SK-N-MC cells with TNF-alpha induces a significant increase in the cell surface expression of CXCR4 in a time- and dose-dependent manner. The effect is partly regulated at the level of transcription. To assess the biological significance of this finding, we show that TNF-alpha potentiates the ability of the above mentioned HIV isolates to induce neuronal apoptosis and that the effect is significantly reduced by pretreating cells with monoclonal antibodies to either CXCR4 and TNF-alpha. Together these results suggest that TNF-alpha may render neuronal cells vulnerable to the apoptotic effects of HIV by increasing the cell surface expression of CXCR4 and thus identify another mechanism by which TNF-alpha contributes to the pathogenesis of HIV-associated brain injury.