Aerobic Biotransformation of N-Nitrosodimethylamine and N-Nitrodimethylamine by Benzene-, Butane-, Methane-, Propane-, and Toluene-Fed Cultures

N-Nitrosodimethylamine (NDMA) is an emerging contaminant of concern. N-nitrodimethylamine (DMNA) is a structural analog to NDMA. NDMA and DMNA have been found in drinking water, groundwater, and other media and are of concern due their toxicity. The authors evaluated biotransformation of NDMA and DMNA by cultures enriched from contaminated groundwater growing on benzene, butane, methane, propane, or toluene. Maximum specific growth rates of enriched cultures on butane (μ_max = 1.1 h^?1) and propane (μ_max = 0.65 h^?1) were 1 to 2 orders of magnitude higher than those presented in the literature. Growth rates of mixed cultures grown on benzene (μ_max = 1.3 h^?1), methane (μ_max = 0.09 h^?1), and toluene (μ_max = 0.99 h^?1) in these studies were similar to those presented in the literature. NDMA biotransformation rates for methane oxidizers (υ_max = 1.4 ng min^?1 mg^?1) and toluene oxidizers (υ_max = 2.3 ng min^?1 mg^?1) were comparable to those presented in the literature, whereas the biotransformation rate for propane oxidizers (υ_max = 0.37 ng min^?1 mg^?1) was lower. NDMA biotransformation rates for benzene oxidizers (υ_max = 1.02 ng min^?1 mg^?1) and butane oxidizers (υ_max = 1.2 ng min^?1 mg^?1) were comparable to those reported for other primary substrates. These studies showed that DMNA biotransformation rates for benzene (υ_max = 0.79 ng min^?1 mg^?1), butane (υ_max = 1.0 ng min^?1 mg^?1), methane (υ_max = 2.1 ng min^?1 mg^?1), propane (υ_max = 1.46 ng min^?1 mg^?1), and toluene (υ_max = 0.52 ng min^?1 mg^?1) oxidizers were all comparable. These studies highlight potential bioremediation methods for NDMA and DMNA in contaminated groundwater.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Hx, a novel fluorescent, minor groove and sequence specific recognition element: design, synthesis, and DNA binding properties of p-anisylbenzimidazole-imidazole/pyrrole-containing polyamides

With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.     

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.     

Flexible Quantitative Risk Assessment for Developmental Toxicity Based on Fractional Polynomial Predictors

Risk assessment for developmental toxicity studies in rodents is faced with the fairly involved data structure of clustered multivariate binary outcomes. While likelihood methods for this setting do not abound, we show that a conditional model, combined with pseudo‐likelihood inference and fractional polynomial predictor functions, as proposed by Royston and Altman (1994), are a promising way forward. The methods are illustrated using teratology data collected under the National Toxicology Program.