Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc.

Metabolism of formycin B by Leishmania amastigotes in vitro. Comparative metabolism in infected and uninfected human macrophages.

Formycin B is metabolized by cutaneous Leishmania amastigotes within cultured human macrophages to give formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates. Formycin A is also incorporated into RNA. The activity of formycin B against amastigotes was correlated with the levels of formycin A metabolites formed in the parasites. Uninfected macrophages also convert formycin B into the same products, but the levels are markedly lower than those seen in infected macrophages. The results suggest that a sufficient therapeutic index exists to warrant consideration of formycin B as an anti-leishmanial drug in humans.

Evolutionary genetics: The economics of mutation.

The presence of mutator genotypes in populations of bacteria may be favoured by selection because they produce rare beneficial mutations and thereby increase the rate of adaptive evolution. Recent work, however, shows that the relationship between mutation rates and adaptive evolution is more complicated.

Adaptive evolution of highly mutable loci in pathogenic bacteria.

Bacteria have specific loci that are highly mutable. We argue that the coexistence within bacterial genomes of such 'contingency' genes with high mutation rates, and 'housekeeping' genes with low mutation rates, is the result of adaptive evolution, and facilitates the efficient exploration of phenotypic solutions to unpredictable aspects of the host environment while minimizing deleterious effects on fitness.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

Bacterial genomics and adaptation to life on plants: implications for the evolution of pathogenicity and symbiosis.

Many bacteria form intimate associations with plants. Despite the agricultural and biotechnological significance of these bacteria, no whole genome sequences have yet been described. Plant-associated bacteria form a phylogenetically diverse group, with representative species from many major taxons. Sequence information from genomes of closely related bacteria, in combination with technological developments in the field of functional genomics, provides new opportunities for determining the origin and evolution of traits that contribute to bacterial fitness and interactions with plant hosts.

The turnover of strains in intermittent and persistent nasal carriers of Staphylococcus aureus.

OBJECTIVES: We aimed to examine the dynamics of Staphylococcus aureus nasal carriage in healthy adults. METHOD: Selected S. aureus strains isolated from weekly nasal swabs obtained from 122 healthy young adults over a 13 week period were spa typed. RESULTS: The median duration of intermittent carriage was 4 weeks (IQR 2-6) and the median interval between episodes of carriage of different spa types was 3.5 weeks (IQR 2.25-4). 6/19 (32%) Persistent carriers were colonised with more than one spa type during the study, and in two persistent carriers a brief period of mixed colonisation with two spa types was observed. Even when the carriage strain changed, it was very rare for persistent carriers to have a period during which they were culture-negative (only 6/188 (3%) swabs submitted by persistent carriers failed to culture S. aureus). CONCLUSIONS: Our results imply that at least every eight weeks a healthy young adult is exposed to S. aureus sufficient to cause a new episode of carriage among intermittent carriers. Persistent carriers are almost always colonised with S. aureus and over the course of a year there will be at least one replacement of the dominant strain.

Detecting linkage disequilibrium in bacterial populations.

The distribution of the number of pairwise differences calculated from comparisons between n haploid genomes has frequently been used as a starting point for testing the hypothesis of linkage equilibrium. For this purpose the variance of the pairwise differences, VD, is used as a test statistic to evaluate the null hypothesis that all loci are in linkage equilibrium. The problem is to determine the critical value of the distribution of VD. This critical value can be estimated either by Monte Carlo simulation or by assuming that VD is distributed normally and calculating a one-tailed 95% critical value for VD, L, L = EVD + 1.645 sqrt(VarVD), where E(VD) is the expectation of VD, and Var(VD) is the variance of VD. If VD (observed) > L, the null hypothesis of linkage equilibrium is rejected. Using Monte Carlo simulation we show that the formula currently available for Var(VD) is incorrect, especially for genetically highly diverse data. This has implications for hypothesis testing in bacterial populations, which are often genetically highly diverse. For this reason we derive a new, exact formula for Var(VD). The distribution of VD is examined and shown to approach normality as the sample size increases. This makes the new formula a useful tool in the investigation of large data sets, where testing for linkage using Monte Carlo simulation can be very time consuming. Application of the new formula, in conjunction with Monte Carlo simulation, to populations of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Bacillus subtilis reveals linkage disequilibrium where linkage equilibrium has previously been reported.

In vivo expression technology strategies: valuable tools for biotechnology.

Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology.

The effect of fluconazole on the clinical pharmacokinetics of methadone.

A randomized, double-blinded, placebo-controlled pharmacokinetic and safety trial was conducted to determine the effect of fluconazole on methadone disposition. Volunteers receiving methadone maintenance therapy were randomized to receive either 200 mg/day oral fluconazole (n = 13) or placebo (n = 12). After 14 days there was a 35% average increase in serum methadone area under the curve relative to baseline among patients receiving fluconazole (p = 0.0008). At the same time, mean serum methadone peak and trough concentrations increased by 27% (p = 0.0076) and 48% (p = 0.0023), respectively, and oral clearance of methadone was reduced by 24% (p = 0.0007). In contrast, the pharmacokinetics of methadone were unaltered in the placebo group. Renal clearance of methadone was not significantly affected by fluconazole or placebo therapy. Although exposed to increased concentrations of methadone, patients treated with fluconazole did not exhibit signs or symptoms of significant narcotic overdose.

A carbon-13 nuclear magnetic resonance analysis of the products of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes.

The major products of glucose metabolism were determined for amastigotes and promastigotes of Leishmania (mexicana) pifanoi under aerobic and anaerobic conditions using carbon-13 nuclear magnetic resonance. Under aerobic conditions, the major products for both forms were carbon dioxide, succinate, malate, acetate and alanine. Succinate was the dominant metabolite of promastigotes, whereas acetate and alanine were most abundant with amastigotes. Under anaerobic conditions, promastigotes produced glycerol as the dominant metabolite, along with lesser amounts of succinate, acetate and alanine; acetate and alanine remained major metabolites in amastigotes, with an increase in the relative amount of succinate and the production of some glycerol. Promastigotes generated carbon dioxide at a 5-fold greater rate than amastigotes under aerobic conditions, but this rate was reduced by more than 95% in the absence of oxygen. Amastigotes were relatively less affected by lack of oxygen and produced carbon dioxide at a rate comparable to promastigotes under anaerobic conditions. The presence of carbohydrates with a possible role in storage was detected in both promastigotes and amastigotes.

Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture.

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.

Screening for lead poisoning in an urban pediatric clinic using samples obtained by fingerstick.

OBJECTIVE: To assess the false positive rate of blood lead (BPb) determinations on samples obtained by fingerstick from children screened in an urban clinic. METHOD: From a single fingerstick (N = 1573), blood was collected in a capillary tube for determining lead concentration (CPb) by graphite furnace and an additional sample was absorbed onto a filter paper for determining lead concentration (FPb) by atomic absorption spectrophotometry with Delves cup. Zinc protoporphyrin (ZPP) was measured immediately and a confirmatory venous lead (VPb) specimen was obtained at the same visit if the ZPP was > or = 35 micrograms/dL (0.6 mumol/L); children with either a CPb or FPb > or = 15 micrograms/dL (0.7 mumol/L) were later recalled for determining VPb. RESULTS: For the 172 children who had a VPb on the same day as the screening tests, the false positive rates (95% confidence intervals) at a lead threshold of 15 micrograms/dL (0.7 mumol/L) were: CPb, 13.5% (6.7-20.3); FPb, 19.1% (11.8-26.4). Analyses using all 679 screens with a paired venous specimen (mean delay between screen and venous testing = 30 days) yielded much higher false positive rates (CPb, 31.3%; FPb, 46.0%). CONCLUSIONS: Screening for lead poisoning is feasible within an urban pediatric clinic by direct measurement of lead concentration in blood samples obtained by fingerstick. The false positive rate that can be obtained is acceptable given the precision of measuring BPb concentration. Practitioners using a staged screening protocol may incorrectly attribute a higher false positive rate to the screening tests, when much of the error may be due to the temporal variability of BPb resulting from both biologic variability in BPb concentration and intermittent exposures.

Management of a Sabiá virus-infected patients in a US hospital.

OBJECTIVE: To describe the hospital precautions used to isolate a Sabiá virus (arenavirus: Arenaviridae)-infected patient in a US hospital and to protect hospital staff and visitors. DESIGN: Investigation of a single case of arenavirus laboratory-acquired infection and associated case-contacts. SETTING: A 900-bed, tertiary-care, university-affiliated medical center. PATIENTS OR OTHER PARTICIPANTS: The case-patient became ill with Sabiá virus infection. The case-contacts consisted of healthcare workers, coworkers, friends, and relatives of the case-patient. INTERVENTION: Enhanced isolation precautions for treatment of a viral hemorrhagic fever (VHF) patient were implemented in the clinical laboratory and patient-care setting to prevent nosocomial transmission. The enhanced precautions included preventing aerosol spread of the virus from the patient or his clinical specimens. All case-contacts were tested for Sabiá virus antibodies and monitored for signs and symptoms of early disease. RESULTS: No cases of secondary infection occurred among 142 case-contacts. CONCLUSIONS: With the frequency of worldwide travel, patients with VHF can be admitted to a local hospital at any time in the United States. The use of enhanced isolation precautions for VHF appeared to be effective in preventing secondary cases by limiting the number of contacts and promoting proper handling of laboratory specimens. Patients with VHF can be managed safely in a local hospital setting, provided that appropriate precautions are planned and implemented.

Effects of digoxin immune Fab (ovine) on digoxin immunoassays.

Digoxin immune Fab (ovine) (Digibind) is a preparation of Fab fragments of antidigoxin antibodies that is used as an antidote for severe digitalis toxicity. To assess the effects of its presence on digoxin immunoassays, specimens were prepared that simulated typical samples from treated patients and were distributed to a number of laboratories for analysis as unknowns. The antidote interfered to a varying extent with all assays reported. The results suggest that most competitive immunoassays will yield results that bear little relation to concentrations of total digoxin or free digoxin (i.e., digoxin not bound to the antidote). The aca, Dimension, and CEDIA sequential immunoassays moderately overestimated the free digoxin concentration. Free digoxin levels appeared to be only slightly overestimated by the Aria, Stratus, and Syva assays. The TDx Digoxin II assay yielded slight to moderate underestimates of the total digoxin concentration.

Identification of patients for pharmacologic review by computer analysis of clinical laboratory drug concentration data.

Clinical pharmacokinetics evaluation and consultation can improve drug therapy and decrease the incidence of adverse reactions in selected patients. However, identification of patients appropriate for review is difficult. The authors developed a microcomputer-based expert system that scans clinical laboratory drug concentration data to identify patients for follow-up. Rules were developed from a review of data for digoxin, phenytoin, and theophylline. These were implemented in software that provides for simple rule creation and modification, on-screen graphic review of data, and printing of chart reports. This program is readily adapted for use with most laboratory information systems. In a retrospective study of 868 patients monitored for digoxin, phenytoin, and theophylline, 29% were flagged as having drug level profiles of possible concern. The majority (62%) of these patients had multiple specimens flagged, suggesting persistent problems. These data suggest that patients can be identified for follow-up by scanning serial drug concentrations, allowing consultative resources to be focused on patients most likely to benefit from them.

Pyrimethamine analysis by enzyme inhibition and HPLC assays.

Pyrimethamine is an antiparasitic agent currently used for therapy of central nervous system toxoplasmosis, a disease seen with increasing frequency in association with the AIDS epidemic. Monitoring of pyrimethamine levels may be particularly important because patients may be treated with high doses of the drug for extended periods of time. The authors have developed and validated both a new enzyme inhibition assay that can be run on an automated analyzer and an improved high performance liquid chromatography (HPLC) method. The calibration range of both methods is 100 to 3,000 micrograms/L. Both demonstrate good linearity, specificity, and precision, and correlate well with one another (r = 0.99). The CVs of the enzyme inhibition assay were < or = 8.6% and those of the HPLC method were < or = 5.4%. No interference was noted for a variety of drugs likely to be used concomitantly with or in lieu of pyrimethamine with the exception of a minor interference from trimethoprim in the enzyme inhibition assay. The major advantage of the enzyme inhibition assay is its ease of automation. The major advantages of the HPLC assay are its precision and relative simplicity. These methods should facilitate therapeutic monitoring of pyrimethamine.

Performance characteristics of three serum iron and total iron-binding capacity methods in acute iron overdose.

Accurate serum iron and total iron binding capacity (TIBC) measurements may be useful in acute iron overdoses. Two alumina column TIBC methods were found to measure increased TIBC when free iron was present. A homogeneous TIBC method gave consistent results until iron concentrations exceeded 500 micrograms/dL (90 mumol/L), when it began to underestimate the TIBC. Serious iron overdoses require chelation therapy with deferoxamine. Iron recovery was reduced by up to 50% for all 3 methods with clinically achievable concentrations of deferoxamine 8,400 micrograms/dL (150 mumol/L). TIBC measurements by both alumina column methods were reduced by deferoxamine in the presence of free iron and unaffected when the iron concentration was less than the TIBC. The homogeneous TIBC method yielded falsely elevated results in the presence of free deferoxamine. Procedures that measure TIBC by addition of excess ferric iron followed by alumina adsorption are not suitable for monitoring TIBC in acute iron overdose. The homogeneous TIBC assay can be used in acute iron overdose but underestimates TIBC when iron concentrations exceed 500 micrograms/dL (90 mumol/L). None of the methods examined are useful for measuring iron or TIBC in the presence of deferoxamine.

Screening for lead poisoning by fingerstick in suburban pediatric practices.

OBJECTIVE: To assess the false-positive rate of blood lead determinations on samples obtained by fingerstick from children screened in private suburban and rural practices. METHODS: Screening capillary lead samples were obtained by fingerstick; children with capillary lead levels of 0.7 mumol/L (15 micrograms/dL) or greater were recalled for a confirmatory venous lead test that served as the criterion standard. Parents completed a five-question risk assessment questionnaire at the time of initial screening. SETTING: Four private suburban to rural practices that serve predominantly white, middle-class populations. PARTICIPANTS: Children seen for routine care between August 1992 and February 1993 (N = 1085; 98% between 6 months and 6 years of age). RESULTS: Capillary lead level was 0.7 mumol/L (15 micrograms/dL) or greater in 35 children (3% of total sample); venous lead samples were obtained in 30 patients. Nine of the elevated capillary lead results were true-positives (venous lead = 0.7, 0.8, 0.8, 0.9, 0.9, 0.9, 1.1, 1.1, and 1.7 mumol/L [15, 17, 17, 18, 18, 18, 22, 22, and 35 micrograms/dL]); parents of only two of these children answered yes to any question on the risk assessment questionnaire. Although the false-positive rate of the capillary lead screening test was 70% (21/30) in this setting, only 2% of the total sample had a false-positive screening test (an average of fewer than one false-positive per month per practice). Screening by fingerstick allowed phlebotomy to be avoided for 97% of the children. CONCLUSION: Fingerstick screening for lead poisoning is a reasonable alternative to direct venous testing within private suburban and rural practices, provided that care is taken to avoid specimen contamination, that appropriate caution is used in the interpretation of screening test results, and that medical and environmental interventions are based on the results of confirmatory venous testing.

Pentavalent antimony-mannan conjugate therapy of experimental visceral leishmaniasis.

Pentavalent antimony-mannan (Sb[V]-mannan) was 10-fold more potent than sodium stibogluconate in a murine model of visceral leishmaniasis. Liver antimony concentrations were six-fold higher after Sb[V]-mannan therapy compared with a dose of sodium stibogluconate that was equipotent in reducing liver parasite burdens. Murine toxicity of Sb[V]-mannan was variable, with a 50% lethal dose (LD50) for one preparation that was well above the concentration that killed 90% of the parasites, and for another preparation was only modestly higher than the concentration that killed 90% of the parasites.