Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Biofilm formation by and thermal niche and virulence characteristics of Escherichia spp

In order to better understand the ecological and virulence characteristics of the various clades of Escherichia, in vitro and in vivo experiments were undertaken. Members of the recently described cryptic clades of Escherichia (clades III, IV, and V) were found to have an enhanced ability to form biofilms compared to strains of Escherichia coli, E. fergusonii, or E. albertii. Members of the cryptic clades were also able to replicate at a lower temperature (5°C versus 11°C) than strains of the named species of Escherichia. Neither a strain's maximal growth rate nor its optimal temperature for growth varied with respect to the strain's phylogenetic affiliation. Escherichia strains not belonging to the species E. coli were positive for a mix of traits thought to enhance a strain's ability to cause either intestinal or extraintestinal disease. However, no non-E. coli Escherichia strain was virulent in a mouse model of extraintestinal infection. The frequency of resistance to antibiotics was low, and none of the strains tested harbored class 1, 2, or 3 integrons. The results of these experiments support the hypothesis that members of the cryptic Escherichia clades may be better able to persist in the external environment compared to E. coli, E. fergusonii, or E. albertii, isolates.     

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.     

Ocean acidification disrupts the innate ability of fish to detect predator olfactory cues

While ocean acidification is predicted to threaten marine biodiversity, the processes that directly impact species persistence are not well understood. For marine species, early life history stages are inherently vulnerable to predators and an innate ability to detect predators can be critical for survival. However, whether or not acidification inhibits predator detection is unknown. Here, we show that newly hatched larvae of the marine fish Amphiprion percula innately detect predators using olfactory cues and this ability is retained through to settlement. Aquarium-reared larvae, not previously exposed to predators, were able to distinguish between the olfactory cues of predatory and non-predatory species. However, when eggs and larvae were exposed to seawater simulating ocean acidification (pH 7.8 and 1000 p.p.m. CO₂) settlement-stage larvae became strongly attracted to the smell of predators and the ability to discriminate between predators and non-predators was lost. Newly hatched larvae were unaffected by CO₂ exposure and were still able to distinguish between predatory and non-predatory fish. If this impairment of olfactory preferences in settlement-stage larvae translates to higher mortality as a result of increased predation risk, there could be direct consequences for the replenishment and the sustainability of marine populations.
Ecology Letters (2010) 13: 68–75     

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.     

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.