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Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been establishedin vitro. While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 (FANCD2-/-) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 (FANCD2-/-) patient cells, includingALDH1A1andRDH10, which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 (FANCD2-/-) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 (FANCD2-/-) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA.

There are limiteddata on long-term changes in the gut microbiome after acute lymphoblastic leukemia (ALL) therapy. We compared the gut microbial composition in stool samples of nine survivors of childhood ALL with 10 healthy sibling controls using 16S rRNA gene sequencing. Analysis of beta diversity within family units demonstrated a significant difference in bacterial strains between patients and healthy siblings. A significant difference in alpha diversity between patients and their healthy siblings was noted using Pielou's evenness. The composition of the gut microbiome differs between pediatric ALL survivors and healthy sibling controls for years after completion of therapy.

Segmented mesocosms (n = 3) packed with sand, sandy loam or clay loam soil were used to determine the effect of soil texture and depth on transport of two septic tank effluent (STE)-borne microbial pathogen surrogates—green fluorescent protein-labeled E. coli (GFPE) and MS-2 coliphage—in soil treatment units. HYDRUS 2D/3D software was used to model the transport of these microbes from the infiltrative surface. Mesocosms were spiked with GFPE and MS-2 coliphage at 105 cfu/mL STE and 105–106 pfu/mL STE, respectively. In all soils, removal rates were >99.99% at 25 cm. The transport simulation compared (1) optimization; and (2) trial-and-error modeling approaches. Only slight differences between the transport parameters were observed between these approaches. Treating both the die-off rates and attachment/detachment rates as variables resulted in an overall better model fit, particularly for the tailing phase of the experiments. Independent of the fitting procedure, attachment rates computed by the model were higher in sandy and sandy loam soils than clay, which was attributed to unsaturated flow conditions at lower water content in the coarser-textured soils. Early breakthrough of the bacteria and virus indicated the presence of preferential flow in the system in the structured clay loam soil, resulting in faster movement of water and microbes through the soil relative to a conservative tracer (bromide).

Although groundwater quality depends on microbial processes in the soil treatment area (STA) of onsite wastewater treatment systems (OWTS), our understanding of the development of these microbial communities is limited. We examined the bacterial communities of sand, sandy loam, and clay STAs at different depths in response to septic tank effluent (STE) addition using mesocosms. Terminal restriction fragment length polymorphism (TRFLP) analysis was used to compare the bacterial community structure and composition of STE, native soil prior to STE addition (UNX) and soil exposed to STE (EXP). Principal component analysis separated communities with depth in sand but not in sandy loam or clay. Indices of richness, diversity, and evenness followed the order: sandy loam > sand > clay. Analysis of TRF peaks indicated that STE contributed least to the composition of STA bacterial communities (5%–16%), followed by UNX soil (18%–48%), with the highest proportion of the community made up of TRFs not detected previously in either UNX or STE (50%–82%) for all three soils. Soil type and depth can have a marked effect on the structure and composition of STA bacterial communities, and on the relative contribution of native soil and STE to these communities.

Antibiotics can be present in low concentrations in domestic wastewater, but little is known about their effect on bacteria in onsite wastewater treatment systems. Mesocosms, consisting of soil-filled lysimeters representing the leachfield of a septic system under aerated (AIR) and unaerated (LEACH) conditions, were used to study the effects of tetracycline addition (5 mg L(-1)) to septic tank effluent on tetracycline resistance in the fecal indicator bacteria Escherichia coli and fecal streptococci, and on their removal. The mesocosms were dosed with antibiotic for 10 days, and effects monitored for 52 days. The fraction of resistant bacteria in mesocosm drainage water relative to that in septic tank effluent, GammaRes, for E. coli ranged from 0 to 0.66 in the AIR treatment and from 0 to 3.32 in the LEACH treatment. For fecal streptococci, GammaRes ranged from 0 to 0.41 and from 0.63 to 1.06 in the AIR and LEACH treatments, respectively. No significant differences in antibiotic resistance of fecal indicator bacteria were observed among sampling dates in soil or water from either treatment. Tetracycline had no significant effect on removal of fecal indicator bacteria, which ranged from 99.9 to 100% for E. coli and from 95.9 to 100% for fecal streptococci. Our results suggest that short-term addition of tetracycline at environmentally-relevant concentrations is likely to have minimal consequences on pathogen removal from wastewater and development of antibiotic resistance among pathogenic bacteria in leachfield soil.

Lyme borreliosis is a vector-borne disease confined primarily to the Northern hemisphere and caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (s.l.) genospecies (Kurtenbach et al. 2006). The spirochetes are maintained exclusively within an enzootic cycle, alternating between a tick vector and vertebrate host. B. burgdorferi s.l. includes three pathogenic species and at least eight related species that are non-pathogenic or rarely cause infection in humans. The three pathogenic species include B. burgdorferi sensu stricto (s.s.), the only species found in North America, and B. garinii and B. afzelii the causative agents of borreliosis in Europe and Asia (Steere et al. 2004). These three species cause similar acute and chronic illness in humans, although regional differences do exist (Steere 2001).

DEC1R06a: Development of Quantitative Tools to Determine the Expected Performance of Unit Process in Wastewater Treatment Units Onsite wastewater treatment system (OWTS) systems are an important part of the wastewater treatment and water management infrastructure in the U.S. Thus, proper OWTS selection, design, installation, operation and management are essential. While OWTS vary widely in their design and implementation, most systems are conventional OWTS that on the soil treatment unit (STU) for wastewater constituent treatment, hydraulic capacity, and eventual recharge to water resources. While there is considerable concern about potential water quality degradation associated with OWTS, current permitting and design focus mainly on ensuring that the hydraulic loading is not excessive. The STU provides an effective and sustainable means for wastewater reclamation, but occasional water quality degradation has been experienced. The likely cause for this is an incomplete understanding of treatment processes in various STUs, and the lack of available tools for assessing the performance of the STU. The overall goal of the project was to provide a toolkit to assess STU performance to enable evaluation and design of expected STU performance for important wastewater constituents over a relevant range of OWTS operating conditions. The toolkit is appropriate for a wide range of users, and includes an implementation protocol for different tools of varying complexity. Specific project objectives were to: 1. identify the best practices, available data, data gaps, and promising tools and techniques utilized in STU design and performance, 2. develop and test tools for performance-based STU design, 3. develop a protocol for using the tools, 4. refine the tools and protocol using data from laboratory studies, field sites, and numerical modeling, and 5. provide a final tool-kit and protocol to aid system designers and decision makers assess the expected STU performance. DEC1R06b: Quantitative Tools to Determine the Expected Performance of Unit Process in Wastewater Treatment Units: Toolkit Users Guide Onsite wastewater treatment systems (OWTS) are an important part of water management infrastructure in the United States. Thus, proper OWTS selection, design, installation, operation and management are essential. To aid this life-cycle, a toolkit was developed to enable evaluation and design of expected STU performance. The toolkit is comprised of this Guidance Manual, a companion Toolkit User's Guide, individual tools, and supplemental information. This framework provides detailed information to less experienced user's while enabling more experienced users to start directly with STUMOD or other tool implementation referring to limited sections of the Guidance Manual or User's Guide. The toolkit was developed for a wide range of users faced with different needs of varying complexity when evaluating treatment of nitrogen, microbial pollutants (bacteria and virus), and organic wastewater contaminants (OWCs). Progressing through simple to more complex tools ultimately guides the user to the simplest tool that is appropriate, but discourages using a tool that is too simple for the decision at hand. The simplest tools include look-up tables and cumulative frequency distributions to direct the user to available pertinent information. Nomographs enable initial screening and quick insight into expected nitrogen removal based on the predicted output from STUMOD. Cumulative probability graphs illustrate modeling results in a risk-based framework while numerical model simulations demonstrate the usefulness of complex tools. Finally, two spreadsheet tools were developed for nitrogen transport, N-CALC and STUMOD, allowing the user to evaluate a range of STU operating conditions, soil hydraulics, and/or treatment parameters, as well as the relative influence of these factors on performance. DEC1R06c: Quantitative Tools to Determine the Expected Performance of Unit Process in Wastewater Treatment Units: Visual-Graphics Tools This file includes the visual-graphic tools: nomographs, cumulative probability graphs, and scenario illustrations. Chapter 1.0 includes nomographs illustrating the fraction of total-nitrogen remaining with depth. Chapter 2.0 includes cumulative probability graphs that illustrate the likely range of treatment outcomes. Chapter 3.0 includes HYDRUS simulation outputs that illustrate various operational scenarios. Finally, a list of visual-graphic tools is provided to aid in locating the visual-graphic tool of interest. This is a separate document that must be used in conjunction with the Guidance Manual and User's Guide. The companion Guidance Manual is organized into four Chapters describing the toolkit and providing guidance for tool selection and use. The fundamental assumptions that were incorporated and a detailed description of the tool development for these visual-graphic tools are provided in the companion User's Guide. Additional tools provided as separate files include STUMOD and N-CALC as MicrosoftTM Xcel files. This title belongs to WERF Research Report Series . In both the nomograph and the cumulative probability graphs, treatment information provided by these tools is based on data generated by numerical models that can incorporate complex and robust treatment and operating conditions. The parameters used for nomograph development are summarized in Table VG-1. Table VG-2 provides a definition for each parameter. Because the choices for representative OWTS conditions are limited, the user must decide how their OWTS system fits within the limited treatment estimations displayed by the graphics. Nomographs and cumulative probability graphs were developed for the following fixed operating conditions: •Effluent Quality ○Standard Effluent = representative of septic tank effluent (STE) as 60 mg-N L-1 as ammonium-nitrogen plus 1 mg-N L-1 as nitrate-nitrogen ○Nitrified Effluent = representative of aerobically treated STE to achieve nitrogen reduction and transformation as 15 mg-N L-1 as nitrate-nitrogen •Hydraulic Loading Rate (HLR) ○2 cm d-1 ○5% Ksat •Regional Temperature Range ○Frigid/Cryic = Average Range 0 to 8 oC, Annual Mean 4.5 oC ○Mesic = Average Range 8 to 15 oC, Annual Mean 11.5 oC ○Thermic = Average Range 15 to 22 oC, Annual Mean 18.5 oC ○Hyperthermic = Average Range 22 to 29 oC, Annual Mean 25.5 oC ISBN: 9781843393955 (eBook)

We evaluated microbiological water quality in the Pettaquamscutt River (Rhode Island, USA), an estuarine river. Fecal coliform (FC) and enterococci (FE) bacteria, presence of Bifidobacterium adolescentis DNA (indicating human fecal contamination), and optical brightener (OB) fluorescence (associated with laundry detergents) were determined for 14 stations from May to September 2010. Six stations had high counts of FE and FC, and the presence of B. adolescentis DNA and high OB fluorescence indicated human fecal contamination - four had septic systems as likely sources of contamination; the others were in sewered areas. The ability of FC and FE to indicate human fecal contamination was assessed against a positive B. adolescentis test. FC and FE had false positive rates of 25% and 17%, respectively, and false negatives of 44% for FC and 63% for FE. Inclusion of molecular and chemical indicators should improve tracking of human fecal contamination sources in the river.

We examined the effects of tetracycline (TET) addition on the function of mesocosms representing aerated and unaerated septic system leachfields. Replicate mesocosms (n = 3) were filled with soil and either vented to a leachfield (LEACH) or aerated intermittently to maintain an O(2) level of approximately 0.21 mol mol(-1) (AIR). All mesocosms were dosed every 6 h for 10 d with 3 cm of domestic wastewater amended with 5 mg TET L(-1). Water quality parameters, headspace gas composition, and soil properties were measured prior to and during the dosing period, and for 42 days after the last antibiotic dose. No significant effect of TET was observed on the pH, level of dissolved O(2) or dissolved organic carbon (DOC) in drainage water from either treatment. In contrast, levels of Fe(2+) and SO(4) in drainage water from LEACH mesocosms decreased in response to TET dosing, with lower levels persisting until Day 52. Persistent increases were observed in the level of NO(3) in drainage water from AIR lysimeters and in NH(4) in LEACH mesocosms in response to TET additions. Removal of total P and DOC were unaffected by TET dosing in either treatment. Nitrogen removal in AIR mesocosms decreased during the TET dosing period, returning to pre-dosing values by Day 52. In contrast, TN removal in LEACH mesocosms increased during TET dosing, returning to pre-dosing values by Day 52. The composition of headspace gases in AIR mesocosms was not affected by tetracycline dosing. TET dosing resulted in significant increases in soil NH(4) concentration in LEACH mesocosms, whereas significant decreases were apparent in AIR mesocosms. Elevated levels of H(2)S and CH(4) in the headspace of LEACH mesocosms coincided with TET dosing and returned to pre-dosing levels when antibiotic dosing ceased. The effects of tetracycline on leachfield mesocosms differed as a function of aeration. Although most effects were transient, with values returning to pre-dosing levels after a 6-week recovery period in both treatments, persistent effects were observed in LEACH mesocosms.

Background The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization. Results Using fluorescent chitinase substrates, we demonstrated an inherent chitinase activity in rabbit serum, a component of the B. burgdorferi growth medium (BSK-II). After inactivating this activity by boiling, we showed that wild-type cells can utilize chitotriose, chitohexose or coarse chitin flakes in the presence of boiled serum and in the absence of free GlcNAc. Further, we replaced the serum component of BSK-II with a lipid extract and still observed growth on chitin substrates without free GlcNAc. In an attempt to knockout B. burgdorferi chitinase activity, we generated mutations in two genes (bb0002 and bb0620) predicted to encode enzymes that could potentially cleave the β-(1,4)-glycosidic linkages found in chitin. While these mutations had no effect on the ability to utilize chitin, a mutation in the gene encoding the chitobiose transporter (bbb04, chbC) did block utilization of chitin substrates by B. burgdorferi. Finally, we provide evidence that chitin utilization in an rpoS mutant is delayed compared to wild-type cells, indicating that RpoS may be involved in the regulation of chitin degradation by this organism. Conclusions The data collected in this study demonstrate that B. burgdorferi can utilize chitin as a source of GlcNAc in the absence of free GlcNAc, and suggest that chitin is cleaved into dimers before being imported across the cytoplasmic membrane via the chitobiose transporter. In addition, our data suggest that the enzyme(s) involved in chitin degradation are at least partially regulated by the alternative sigma factor RpoS.