Real-world NOx emissions from heavy-duty diesel, natural gas, and diesel hybrid electric vehicles of different vocations on California roadways.

This study assessed the real-world nitrogen oxide (NOx) emissions from 50 heavy-duty vehicles of different vocations and engine technologies using portable emissions measurement systems (PEMS). This is one of the most comprehensive in-use emissions studies conducted to date, which played a key role in the development of CARB's (California Air Recourses Board) updated EMission FACtor (EMFAC) model, especially for natural gas vehicles. In-use emissions testing was performed on school and transit buses, refuse haulers, goods movement vehicles, and delivery vehicles while were driven over their normal operating routes in the South Coast Air Basin. Engine technologies included diesel engines with and without selective catalytic reduction (SCR) systems, compressed natural gas (CNG) engines and liquified petroleum gas (LPG) engines, and SCR-equipped diesel hybrid electric vehicles. For most vehicles, the in-use NOx emissions were higher than the certification standards for the engine. Diesel vehicles generally showed higher brake-specific NOx emissions compared to the CNG vehicles. NOx emissions were strongly dependent on the SCR temperature, with SCR temperatures below 200 °C resulting in elevate brake-specific NOx. The 0.02 g/bhp-hr certified CNG vehicles showed the largest reductions in NOx emissions. The diesel hybrid electric vehicles showed important distance-specific NOx benefits compared to the conventional diesel vehicles, but higher emissions compared to the CNG and LPG vehicles. Overall, average NOx reductions were 75%, 94%, 65%, 79%, respectively, for the 0.2 CNG, 0.02 CNG, diesel hybrid electric, and LPG vehicles compared to diesel vehicles, due in part to some diesel vehicles with particularly high emissions, indicating that the widespread implementation of advanced technology and alternative fuel vehicles could provide important NOx reductions and a path for meeting air quality targets in California and elsewhere.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells.

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

In vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a Gram-negative capnophilic rod and the etiological agent of localized aggressive periodontitis. The genome-wide survey of A. actinomycetemcomitans using in vivo induced antigen technology (IVIAT) has previously resulted in the discovery of antigenic determinants expressed specifically in diseased patients. The present study evaluated the potential of these antigens as putative disease markers, and investigating their contribution to the pathogenesis of the microorganism. Sera from patients had a significantly greater antibody titer than sera from healthy controls against six antigens, which supports the in vivo expression of these antigens, and suggests their usefulness as disease markers. A. actinomycetemcomitans invasion of epithelium-derived HeLa cells resulted in the induction of all three genes tested, as evidenced by real-time PCR. Isogenic mutants of these three genes were constructed and the adhesion and intracellular survival of the mutants was assayed in a competition assay with the wild-type strain. A significant defect in the intracellular survival of two of these mutant strains (orf1402 and orf859) was found. This defect could not be attributed to an adhesion defect. In contrast, a mutation in vapA, a homologue of a novel putative transcriptional regulator, out-competed the wild-type strain in the same assay. The virulent phenotype was restored for a mutant strain in orf859 upon complementation. This data provided new insight into the pathogenic personality of A. actinomycetemcomitans in vivo and supported the use of HeLa cells as a valid in vitro host-pathogen interactions model for that microorganism. IVIAT is applicable to most pathogens and will undoubtedly lead to the discovery of novel therapies, antibiotics and diagnostic tools.

Genes of periodontopathogens expressed during human disease.

BACKGROUND: Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. METHODS: A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. RESULTS: One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. CONCLUSION: MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.