A ΔdinB mutation that sensitizes Escherichia coli to the lethal effects of UV- and X-radiation.

The DinB (PolIV) protein of Escherichia coli participates in several cellular functions. We investigated a dinB mutation, Δ(dinB-yafN)883(::kan) [referred to as ΔdinB883], which strongly sensitized E. coli cells to both UV- and X-radiation killing. Earlier reports indicated dinB mutations had no obvious effect on UV radiation sensitivity which we confirmed by showing that normal UV radiation sensitivity is conferred by the ΔdinB749 allele. Compared to a wild-type strain, the ΔdinB883 mutant was most sensitive (160-fold) in early to mid-logarithmic growth phase and much less sensitive (twofold) in late log or stationary phases, thus showing a growth phase-dependence for UV radiation sensitivity. This sensitizing effect of ΔdinB883 is assumed to be completely dependent upon the presence of UmuDC protein; since the ΔdinB883 mutation did not sensitize the ΔumuDC strain to UV radiation killing throughout log phase and early stationary phase growth. The DNA damage checkpoint activity of UmuDC was clearly affected by ΔdinB883 as shown by testing a umuC104 ΔdinB883 double-mutant. The sensitivities of the ΔumuDC strain and the ΔdinB883 ΔumuDC double-mutant strain were significantly greater than for the ΔdinB883 strain, suggesting that the ΔdinB883 allele only partially suppresses UmuDC activity. The ΔdinB883 mutation partially sensitized (fivefold) uvrA and uvrB strains to UV radiation, but did not sensitize a ΔrecA strain. A comparison of the DNA sequences of the ΔdinB883 allele with the sequences of the Δ(dinB-yafN)882(::kan) and ΔdinB749 alleles, which do not sensitize cells to UV radiation, revealed ΔdinB883 is likely a "gain-of-function" mutation. The ΔdinB883 allele encodes the first 54 amino acids of wild-type DinB followed by 29 predicted residues resulting from the continuation of the dinB reading frame into an adjacent insertion fragment. The resulting polypeptide is proposed to interfere directly or indirectly with UmuDC function(s) involved in protecting cells against the lethal effects of radiation.

Warming intravenous fluids for improved patient comfort in the emergency department: a pilot crossover randomized controlled trial.

INTRODUCTION: The purpose of this study was to test if intravenous (IV) fluids warmed to body temperature are associated with greater patient comfort than room temperature IV fluids in adult emergency department (ED) patients. METHODS: This was a pilot double-blind, crossover, randomized controlled trial. Enrolled subjects sequentially received boluses of body temperature (36°C) and room temperature (22 °C) IV fluid, with the order of boluses randomized. Each subject's level of discomfort was assessed prior to and after each bolus, using a 10 cm visual analog scale (Discomfort VAS), with higher scores indicating greater discomfort. We calculated the change in Discomfort VAS score associated with body temperature IV fluid (ΔVASbody) and room temperature IV fluid (ΔVASroom) by subtracting the score reported before the bolus from the score reported after that bolus. We compared changes in Discomfort VAS score with body temperature and room temperature IV fluid using the Wilcoxon matched-pairs signed-rank test. RESULTS: Twenty-seven subjects were included. Treatment with body temperature IV fluid was associated with a significant decrease in discomfort (median ΔVASbody: -0.7 cm; interquartile range (IQR): -4.5 cm to +0.4 cm) compared to room temperature IV fluid (median ΔVASroom: +1.2 cm; interquartile range: -0.1 cm to + 3.6 cm) (P = 0.001). CONCLUSION: In this small trial of adult ED patients, infusing IV fluids warmed to body temperature was associated with improved comfort compared to standard, room temperature IV fluids.

Extracellular matrix proteins and astrocyte-derived soluble factors influence the differentiation and proliferation of adult neural progenitor cells.

To control the differentiation of neural progenitor cells (NPCs), the synergistic influence of topography, extracellular matrix (ECM) proteins, and soluble factors were investigated. Previously, in our laboratory, astrocyte-derived soluble factors were found to promote differentiation of adult hippocampal progenitor cells (AHPCs) into neurons when grown on a laminin substrate (Oh et al., J Biomed Mater Res A 2009;91:575-585). Here, we determined that the ECM protein on which AHPCs are cultured does not seem to alter this neurogenic effect or the differentiation of AHPCs when grown alone. However, AHPCs cultured on ECL (a combination of entactin, collagen, and laminin) in the presence of soluble factors from hippocampal astrocytes, differentiated into a significantly greater percentage of oligodendrocytes (approximately 34% on ECL vs. approximately 19% on laminin). Furthermore, a concomitant decrease in the percentage of proliferating cells was observed on the ECL (approximately 38% on ECL vs. approximately 55% on laminin). In addition, the increase in AHPC differentiation into oligodendrocytes on ECL occurred only in the presence of soluble factors from astrocytes, and not when AHPCs were cultured alone. Finally, we demonstrated that micro-scale topography did not influence the phenotypic differentiation in all conditions tested. These results show that a combination of astrocyte-derived soluble factors and ECM can dramatically affect the differentiation and proliferation of NPCs.

A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli.

Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincorporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the alpha subunit of tryptophan synthase.

Suprapubic bladder tap aspiration in an elderly female.

Suprapubic bladder aspiration is a rarely used technique for obtaining urine samples in the adult Emergency Department (ED). We report a case of an elderly woman in whom suprapubic aspiration (SPA) was safely performed using bedside ultrasound guidance. More conventional urine collection methods were precluded by the constraint on positioning imposed by the patient's flexion contractures. Ultrasound-guided SPA is a safe technique, that probably should be employed more frequently in the adult ED.

Interactions among the Escherichia coli mutT, mutM, and mutY damage prevention pathways.

We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis. mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases. To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles. We report three major findings. First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds. We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A. Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP. Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.