Differential Regulation of Retinoic Acid Metabolism in Fanconi Anemia.

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 established in 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, including ALDH1A1 and RDH10 , 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.

Evaluation of microbiological water quality in the Pettaquamscutt River (Rhode Island, USA) using chemical, molecular and culture-dependent methods.

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.

The chitobiose transporter, chbC, is required for chitin utilization in Borrelia burgdorferi.

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 beta-(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.

Effects of tetracycline on water quality, soil and gases in aerated and unaerated leachfield mesocosms.

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.

Effects of tetracycline on antibiotic resistance and removal of fecal indicator bacteria in aerated and unaerated leachfield mesocosms.

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.