Assessment of Non-Occupational 1,4-Dioxane Exposure Pathways from Drinking Water and Product Use.

1,4-Dioxane is a persistent and mobile organic chemical that has been found by the United States Environmental Protection Agency (USEPA) to be an unreasonable risk to human health in some occupational contexts. 1,4-Dioxane is released into the environment as industrial waste and occurs in some personal-care products as an unintended byproduct. However, limited exposure assessments have been conducted outside of an occupational context. In this study, the USEPA simulation modeling tool, Stochastic Human Exposure and Dose Simulator-High Throughput (SHEDS-HT), was adapted to estimate the exposure and chemical mass released down the drain (DTD) from drinking water consumption and product use. 1,4-Dioxane concentrations measured in drinking water and consumer products were used by SHEDS-HT to evaluate and compare the contributions of these sources to exposure and mass released DTD. Modeling results showed that compared to people whose daily per capita exposure came from only products (2.29 × 10-7 to 2.92 × 10-7 mg/kg/day), people exposed to both contaminated water and product use had higher per capita median exposures (1.90 × 10-6 to 4.27 × 10-6 mg/kg/day), with exposure mass primarily attributable to water consumption (75-91%). Last, we demonstrate through simulation that while a potential regulatory action could broadly reduce DTD release, the proportional reduction in exposure would be most significant for people with no or low water contamination.

Contributions of 5'-UTR and 3'-UTR cis elements to Cyt1Aa synthesis in Bacillus thuringiensis subsp. israelensis.

The biopesticide used most effectively to control mosquito and blackfly vectors of human diseases worldwide is Bacillus thuringiensis subsp. israelensis. The high efficacy of this bacterium is due to synergistic interactions among four protein entomotoxins assembled individually into a single parasporal body (PB) during sporulation. Cyt1Aa, the primary synergist, is the most abundant toxin, comprising approximately 55% of the PB's mass. The other proteins are Cry11Aa at ∼35%, and Cry4Aa and Cry4Ba, which together account for the remaining ∼10%. The molecular genetic basis for the comparatively large amount of Cyt1Aa synthesized is unknown. Here, in addition to the known strong BtI (σE) and BtII (σK) promoters, we demonstrate a third promoter (BtIII) that has high identity to the σE promoter of Bacillus subtilis, contributes to the large amount of Cyt1Aa synthesized. We also show that a cyt1Aa-BtIII construct was not functional in a σE-deficient strain of B. subtilis. Comparison of transcription levels and protein profiles for recombinant strains containing different combinations of BtI, BtII and BtIII, or each promoter alone, showed that BtIII is active throughout sporulation. We further demonstrate that a stable stem-loop in the 3'-untranslated region (3'-UTR, predicted ΔG=-27.6) contributes to the high level of Cyt1Aa synthesized.

A 1.1-kilobase region downstream of the bin operon in Bacillus sphaericus strain 2362 decreases bin yield and crystal size in strain 2297.

The 2297 strain of Bacillus sphaericus produces a crystal of the Bin (binary) toxin that is approximately fourfold larger than that of strain 2362, the strain currently used in VectoLex, a commercial mosquito larvicide. Comparison of the regions downstream from the bin operon in these two strains showed that strain 2362 contained a 1.6-kb region with four orf genes not found in strain 2297. Insertion of a 1.1-kb portion of this region from strain 2362 by homologous recombination downstream from the bin operon in strain 2297 reduced Bin toxin production by 50 to 70% and toxicity to fourth-instar larvae of Culex quinquefasciatus by 68%. These results suggest that the 1.6-kb region downstream from the bin operon in B. sphaericus 2362 is responsible for the lower Bin yield and smaller crystal size characteristic of this strain.

Developing recombinant bacteria for control of mosquito larvae.

Genetic engineering techniques have been used to significantly improve mosquito larvicides based on the bacteria Bacillus thuringiensis (Bt) subsp. israelensis (Bti) and Bacillus sphaericus (Bs). These new larvicides hold excellent promise for providing better and more cost-effective control of nuisance mosquitoes and vectors of important diseases, including the anopheline vectors of malaria and culicine vectors responsible for filariasis and viral encephalitides. The toxicity of Bti and Bs is due primarily to endotoxin proteins produced during sporulation. After ingestion by larvae, these are activated and destroy the larval stomach, quickly resulting in death. By cloning the genes encoding various endotoxins from Bt and Bs species, and engineering these for high levels of synthesis, we have been able to generate recombinant bacterial strains based on Bti that are more than 10 times as effective as the conventional strains of Bti or Bs that serve as the active ingredients of commercial bacterial larvicides currently used for mosquito control. The best of these recombinants contain all major Bti endotoxins, specifically, Cry4A, Cry4B, Cry11A, and Cyt1A, plus the binary (Bin) endotoxin of Bs, the principal mosquitocidal protein responsible for the activity of this species. The presence of Cyt1A in these recombinants, which synergizes Cry toxicity and delays resistance to these proteins and Bs Bin, should enable long term use of these recombinants with little if any development of resistance. In the field, these new recombinants should be particularly effective larvicides against most important vectors and nuisance species of the genus Culex, the malaria vectors Anopheles gambiae and An. arabiensis, and species of Aedes and Ochlerotatus sensitive to Bs.

Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.

The nematode Caenorhabditis elegans is an important model for studying the genetics of ageing, with over 50 life-extension mutations known so far. However, little is known about the pathobiology of ageing in this species, limiting attempts to connect genotype with senescent phenotype. Using ultrastructural analysis and visualization of specific cell types with green fluorescent protein, we examined cell integrity in different tissues as the animal ages. We report remarkable preservation of the nervous system, even in advanced old age, in contrast to a gradual, progressive deterioration of muscle, resembling human sarcopenia. The age-1(hx546) mutation, which extends lifespan by 60-100%, delayed some, but not all, cellular biomarkers of ageing. Strikingly, we found strong evidence that stochastic as well as genetic factors are significant in C. elegans ageing, with extensive variability both among same-age animals and between cells of the same type within individuals.

Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support.

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.