APDS: the autonomous pathogen detection system

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.     

Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK

Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Marine actinomycetes related to the "Salinospora" group from the Great Barrier Reef sponge Pseudoceratina clavata

Ten strains identified as marine actinomycetes related to the "Salinospora" group previously reported only from marine sediments were isolated from the Great Barrier Reef marine sponge Pseudoceratina clavata. The relationship of the isolates to "Salinospora" was confirmed by phylogenetic analysis of 16S rRNA gene sequences. Colony morphology and pigmentation, occurrence and position of spores, and salinity requirements for growth were all consistent with this relationship. Genes homologous to beta-ketosynthase, an enzyme forming part of a polyketide synthesis complex, were retrieved from these isolates; these genes shared homology with other Type I ketosynthase genes, and phylogenetic comparison with amino acid sequences derived from database beta-ketosynthase genes was consistent with the close relationship of these isolates to the actinomycetes. Primers based on 16S rRNA gene sequences and designed for targeting amplification of members of the "Salinospora" group via polymerase chain reaction have been used to demonstrate occurrence of these actinomycetes within the sponge tissue. In vitro bioassays of extracts from the isolates for antibiotic activity demonstrated that these actinomycetes have the potential to inhibit other sponge symbionts in vivo, including both Gram-negative and Gram-positive bacteria.     

Correlates of delayed disease progression in HIV-1-infected Kenyan children

Without treatment most HIV-1-infected children in Africa die before their third birthday (>89%) and long-term nonprogressors are rare. The mechanisms underlying nonprogression in HIV-1-infected children are not well understood. In the present study, we examined potential correlates of delayed HIV disease progression in 51 HIV-1-infected African children. Children were assigned to progression subgroups based on clinical characterization. HIV-1-specific immune responses were studied using a combination of ELISPOT assays, tetramer staining, and FACS analysis to characterize the magnitude, specificity, and functional phenotype of HIV-1-specific CD8(+) and CD4(+) T cells. Host genetic factors were examined by genotyping with sequence-specific primers. HIV-1 nef gene sequences from infecting isolates from the children were examined for potential attenuating deletions. Thymic output was measured by T cell rearrangement excision circle assays. HIV-1-specific CD8(+) T cell responses were detected in all progression groups. The most striking attribute of long-term survivor nonprogressors was the detection of HIV-1-specific CD4(+) Th responses in this group at a magnitude substantially greater than previously observed in adult long-term nonprogressors. Although long-term survivor nonprogressors had a significantly higher percentage of CD45RA(+)CD4(+) T cells, nonprogression was not associated with higher thymic output. No protective genotypes for known coreceptor polymorphisms or large sequence deletions in the nef gene associated with delayed disease progression were identified. In the absence of host genotypes and attenuating mutations in HIV-1 nef, long-term surviving children generated strong CD4(+) T cell responses to HIV-1. As HIV-1-specific helper cells support anti-HIV-1 effector responses in active disease, their presence may be important in delaying disease progression.