Perspective on Improving Environmental Monitoring of Biothreats.

For more than a decade, the United States has performed environmental monitoring by collecting and analyzing air samples for a handful of biological threat agents (BTAs) in order to detect a possible biological attack. This effort has faced numerous technical challenges including timeliness, sampling efficiency, sensitivity, specificity, and robustness. The cost of city-wide environmental monitoring using conventional technology has also been a challenge. A large group of scientists with expertise in bioterrorism defense met to assess the objectives and current efficacy of environmental monitoring and to identify operational and technological changes that could enhance its efficacy and cost-effectiveness, thus enhancing its value. The highest priority operational change that was identified was to abandon the current concept of city-wide environmental monitoring because the operational costs were too high and its value was compromised by low detection sensitivity and other environmental factors. Instead, it was suggested that the focus should primarily be on indoor monitoring and secondarily on special-event monitoring because objectives are tractable and these operational settings are aligned with likelihood and risk assessments. The highest priority technological change identified was the development of a reagent-less, real-time sensor that can identify a potential airborne release and trigger secondary tests of greater sensitivity and specificity for occasional samples of interest. This technological change could be transformative with the potential to greatly reduce operational costs and thereby create the opportunity to expand the scope and effectiveness of environmental monitoring.

Microbial community profiling of human saliva using shotgun metagenomic sequencing.

Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS) is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn) analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUS and BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples.

5-Deazaflavin derivatives as inhibitors of p53 ubiquitination by HDM2.

Based on previous reports of certain 5-deazaflavin derivatives being capable of activating the tumour suppressor p53 in cancer cells through inhibition of the p53-specific ubiquitin E3 ligase HDM2, we have conducted an structure-activity relationship (SAR) analysis through systematic modification of the 5-deazaflavin template. This analysis shows that HDM2-inhibitory activity depends on a combination of factors. The most active compounds (e.g., 15) contain a trifluoromethyl or chloro substituent at the deazaflavin C9 position and this activity depends to a large extent on the presence of at least one additional halogen or methyl substituent of the phenyl group at N10. Our SAR results, in combination with the HDM2 RING domain receptor recognition model we present, form the basis for the design of drug-like and potent activators of p53 for potential cancer therapy.

Small molecules that bind the Mdm2 RING stabilize and activate p53.

p53 is a tumor suppressor that responds to a variety of stresses such as oncogenes and DNA damage by activating its transcriptional targets to allow repair or elimination of damaged cells. In the absence of stress signals, p53 needs to be kept in check and this is achieved by the E3 ligase MDM2. For tumors that retain wild-type p53, therapeutic strategies aimed at removing the inhibitory activity of MDM2 on p53 are under development and to date have focused on drugs that prevent the binding of p53 to MDM2. Here, we report the analysis of a group of synthetic analogs derived from 5-deazaflavin compounds previously identified in a screen as inhibitors of MDM2 autoubiquitination. Using measurement of surface plasmon resonance, we demonstrated that active 5-deazaflavin analogs bind to the MDM2 RING, whereas inactive compounds show no binding. In cellular assays, these active MDM2 RING binding compounds inhibited the ubiquitination of p53, stabilized p53, led to increased expression of p53 targets and caused corresponding cell cycle effects. Deazaflavin analogs therefore function to activate p53 through a novel mechanism, by inhibiting the E3 ligase activity of MDM2 in a manner that involves binding to the MDM2 RING.

Short-read, high-throughput sequencing technology for STR genotyping.

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples.

Small-molecule inhibitors of MDM2 as new anticancer therapeutics.

It has long been known that traditional anticancer radio- and chemotherapies in part work through direct or indirect activation of the p53 tumour suppressor pathway. However, many of these strategies are nonselective and genotoxic. The emerging understanding of the pathways that regulate p53 has led to the notion that it should be possible to activate the p53 pathway in ways that are inherently nongenotoxic. Important targets for pharmacological interference in this respect are MDM2 and MDMX, key negative regulators of p53. Genetic and pharmacologic studies suggest that blocking the physical interaction of these proteins with p53, or inhibiting the catalytic role of MDM2 in tagging p53 for proteasomal degradation, both of which lead to an increase in the transcriptional activity of p53, may indeed be an efficient and safe way to eradicate tumour cells that retain wild-type p53. Here we review the rationale for such strategies, as well as the current state in the discovery and development of drugs that reactivate p53 by inhibiting its inhibitors MDM2 and MDMX. The first compounds that have been shown in model systems to be able selectively to kill cancer cells in this way are now entering clinical trials and the promise of MDM2 inhibitors as a new therapeutic anticancer modality should therefore become clear in the not-too-distant future.

Fascaplysin-inspired diindolyls as selective inhibitors of CDK4/cyclin D1.

We present the design, synthesis and biological activity of a new series of substituted 3-(2-(1H-indol-1-yl)ethyl)-1H-indoles and 1,2-di(1H-indol-1-yl)alkanes as selective inhibitors of CDK4/cyclin D1. The compounds were designed to explore the relationship between the connection mode of the indolyl moieties and their CDK inhibitory activities. We found all the above-mentioned designed compounds to be selective inhibitors of CDK4/cyclin D1 compared to the closely related CDK2/cyclin A, with IC(50) for the best compounds 10m and 13a being 39 and 37microm, respectively.