Value-based comparison of ambulatory children with respiratory diseases in an emergency department and a walk-in clinic: a retrospective cohort study in Québec, Canada.

OBJECTIVE: To compare health outcomes and costs given in the emergency department (ED) and walk-in clinics for ambulatory children presenting with acute respiratory diseases. DESIGN: A retrospective cohort study. SETTING: This study was conducted from April 2016 to March 2017 in one ED and one walk-in clinic. The ED is a paediatric tertiary care centre, and the clinic has access to lab tests and X-rays. PARTICIPANTS: Inclusion criteria were children: (1) aged from 2 to 17 years old and (2) discharged home with a diagnosis of upper respiratory tract infection (URTI), pneumonia or acute asthma. MAIN OUTCOME MEASURES: The primary outcome measure was the proportion of patients returning to any ED or clinic within 3 and 7 days of the index visit. The secondary outcome measures were the mean cost of care estimated using time-driven activity-based costing and the incidence of antibiotic prescription for URTI patients. RESULTS: We included 532 children seen in the ED and 201 seen in the walk-in clinic. The incidence of return visits at 3 and 7 days was 20.7% and 27.3% in the ED vs 6.5% and 11.4% in the clinic (adjusted relative risk at 3 days (aRR) (95% CI) 3.17 (1.77 to 5.66) and aRR at 7 days 2.24 (1.46 to 3.44)). The mean cost (95% CI) of care (CAD) at the index visit was $C96.68 (92.62 to 100.74) in the ED vs $C48.82 (45.47 to 52.16) in the clinic (mean difference (95% CI): 46.15 (41.29 to 51.02)). Antibiotic prescription for URTI was less common in the ED than in the clinic (1.5% vs 16.4%; aRR 0.10 (95% CI 0.03 to 0.32)). CONCLUSIONS: The incidence of return visits and cost of care were significantly higher in the ED, while antibiotic use for URTI was more frequent in the walk-in clinic. These data may help determine which setting offers the highest value to ambulatory children with acute respiratory conditions.

A value-based comparison of the management of respiratory diseases in walk-in clinics and emergency departments.

OBJECTIVES: Our aim was to compare some of the health outcomes and costs associated with value of care in emergency departments (ED) and walk-in clinics for ambulatory patients presenting with an acute respiratory disease. METHODS: A health records review was conducted from April 2016 through March 2017 in one ED and one walk-in clinic. Inclusion criteria were: (i) ambulatory patients at least 18 years old, (ii) discharged home with a diagnosis of upper respiratory tract infection (URTI), pneumonia, acute asthma, or acute exacerbation of chronic obstructive pulmonary disease. Primary outcome was the proportion of patients returning to any ED or walk-in clinic within three and seven days of the index visit. Secondary outcomes were the mean cost of care and the incidence of antibiotic prescription for URTI patients. The cost of care was estimated from the Ministry of Health's perspectives using time-driven activity-based costing. RESULTS: The ED group included 170 patients and the walk-in clinic group 326 patients. The return visit incidences at three and seven days were, respectively, 25.9% and 38.2% in the ED vs. 4.9% and 14.7% in the walk-in clinic (adjusted relative risk (arr) of 4.7 (95% CI 2.6-8.6) and 2.7 (1.9-3.9)). The mean cost ($Cdn) of the index visit care was 116.0 (106.3-125.7) in the ED vs. 62.5 (57.7-67.3) in the walk-in clinic (mean difference of 56.4 (45.7-67.1)). Antibiotic prescription for URTI was 5.6% in the ED vs. 24.7% in the walk-in clinic (arr 0.2, 0.01-0.6). CONCLUSIONS: This study is the first in a larger research program to compare the value of care between walk-in clinics and the ED. The potential advantages of walk-in clinics over EDs (lower costs, lower incidence of return visits) for ambulatory patients with respiratory diseases should be considered in healthcare planning.

RéSUMé: OBJECTIFS: Notre objectif était de comparer certains des résultats sanitaires et des coûts associés à la valeur des soins dans les services d'urgence et les cliniques sans rendez-vous pour les patients ambulatoires souffrant d'une maladie respiratoire aiguë. MéTHODES: Une revue des dossiers médicaux a été réalisée d'avril 2016 à mars 2017 dans un service d'urgence et une clinique sans rendez-vous. Les critères d'inclusion étaient les suivants : (i) patients ambulatoires âgés d'au moins 18 ans, (ii) renvoyés chez eux avec un diagnostic d'infection des voies respiratoires supérieures (IVRS), de pneumonie, d'asthme aigu ou d'exacerbation aiguë de la maladie pulmonaire obstructive chronique. Le résultat primaire était la proportion de patients retournant à un service d'urgence ou à une clinique sans rendez-vous dans les trois et sept jours suivant la visite de référence. Les résultats secondaires étaient le coût moyen des soins et l'incidence de la prescription d'antibiotiques pour les patients atteints d'IVRS. Le coût des soins a été estimé à partir des perspectives du ministère de la santé, en utilisant la méthode de calcul des coûts par activité en fonction du temps. RéSULTATS: Le groupe des urgences comprenait 170 patients et le groupe des cliniques sans rendez-vous 326 patients. Les incidences des visites de retour à trois et sept jours étaient respectivement de 25,9 % et 38,2 % dans le service des urgences contre 4,9 % et 14,7 % à la clinique sans rendez-vous (risque relatif ajusté (arr) de 4,7 (IC à 95 % 2,6 à 8,6) et 2,7 (1,9-3,9)). Le coût moyen ($CAN) de la visite de référence était de 116,0 (106,3-125,7) aux urgences contre 62,5 (57,7-67,3) dans la clinique sans rendez-vous (différence moyenne de 56,4 (45,7-67,1)). La prescription d'antibiotiques pour l'IVRS était de 5,6 % aux urgences contre 24,7 % dans la clinique sans rendez-vous (arr 0,2, 0,01-0,6). CONCLUSIONS: Cette étude est la première d'un programme de recherche plus vaste visant à comparer la valeur des soins entre les cliniques sans rendez-vous et les urgences. Les avantages potentiels des cliniques sans rendez-vous par rapport aux services d'urgence (coûts moindres, incidence plus faible des visites de retour) pour les patients ambulatoires souffrant de maladies respiratoires devraient être pris en compte dans la planification des soins de santé.

Adaptation of time-driven activity-based costing to the evaluation of the efficiency of ambulatory care provided in the emergency department.

Objectives: The aim of this study was: (1) to adapt the time-driven activity-based costing (TDABC) method to emergency department (ED) ambulatory care; (2) to estimate the cost of care associated with frequently encountered ambulatory conditions; and (3) to compare costs calculated using estimated time and objectively measured time. Methods: TDABC was applied to a retrospective cohort of patients with upper respiratory tract infections, urinary tract infections, unspecified abdominal pain, lower back pain and limb lacerations who visited an ED in Québec City (Canada) during fiscal year 2015-2016. The calculated cost of care was the product of the time required to complete each care procedure and the cost per minute of each human resource or equipment involved. Costing based on durations estimated by care professionals were compared to those based on objective measurements in the field. Results: Overall, 220 care episodes were included and 3080 time measurements of 75 different processes were collected. Differences between costs calculated using estimated and measured times were statistically significant for all conditions except lower back pain and ranged from $4.30 to $55.20 (US) per episode. Differences were larger for conditions requiring more advanced procedures, such as imaging or the attention of ED professionals. Conclusions: The greater the use of advanced procedures or the involvement of ED professionals in the care, the greater is the discrepancy between estimated-time-based and measured-time-based costing. TDABC should be applied using objective measurement of the time per procedure.

Cardiac Implantable Electronic Device Infection: Detailed Analysis of Cost Implications.

BACKGROUND: Infections of cardiac implantable electronic devices (CIED) are associated with significant morbidity and mortality. Despite many preventive measures, this condition is associated with significant costs for the health care system. METHODS: We retrospectively analyzed all infection cases referred for lead extraction at a single university hospital over 1 year (2015-2016). We then calculated all costs related to the infection episode per patient using hospital databases and charts review. RESULTS: Thirty-eight patients with CIED infections (29% women-mean age 71 ± 14 years) were referred for lead extraction (27 pocket infections, 11 endocarditis). Devices were mainly pacemakers (60%). When the pathogen was identified, Staphylococcus aureus methicillin sensitive was the main cause. Extraction was performed in all but 3 cases (92%). One death occurred in the nonextracted group. Respective durations of hospitalization and intravenous and antibiotic administration for patients undergoing extraction were 21 and 36 days. The calculated mean total cost for CIED infection management was CAD$29,907 (median: 26,879; range: CAD$4,827-$62,585). Mean hospital charges were CAD$12,291, accounting for 41% of the total costs. CONCLUSIONS: This study represents the first analysis of the direct costs associated with lead extraction in Canada. Device infections are associated with significant costs and increased morbidity. Any preventive measure will have a significant impact on the economic burden of the health care system and patient outcome after lead extraction.

Cost-effectiveness of FreeO2 in patients with chronic obstructive pulmonary disease hospitalised for acute exacerbations: analysis of a pilot study in Quebec.

OBJECTIVE: Conduct a cost-effectiveness analysis of FreeO2 technology versus manual oxygen-titration technology for patients with chronic obstructive pulmonary disease (COPD) hospitalised for acute exacerbations. SETTING: Tertiary acute care hospital in Quebec, Canada. PARTICIPANTS: 47 patients with COPD hospitalised for acute exacerbations. INTERVENTION: An automated oxygen-titration and oxygen-weaning technology. METHODS AND OUTCOMES: The costs for hospitalisation and follow-up for 180 days were calculated using a microcosting approach and included the cost of FreeO2 technology. Incremental cost-effectiveness ratios (ICERs) were calculated using bootstrap resampling with 5000 replications. The main effect variable was the percentage of time spent at the target oxygen saturation (SpO2). The other two effect variables were the time spent in hyperoxia (target SpO2+5%) and in severe hypoxaemia (SpO2 <85%). The resamplings were based on data from a randomised controlled trial with 47 patients with COPD hospitalised for acute exacerbations. RESULTS: FreeO2 generated savings of 20.7% of the per-patient costs at 180 days (ie, -$C2959.71). This decrease is nevertheless not significant at the 95% threshold (P=0.13), but the effect variables all improved (P<0.001). The improvement in the time spent at the target SpO2 was 56.3%. The ICERs indicate that FreeO2 technology is more cost-effective than manual oxygen titration with a savings of -$C96.91 per percentage point of time spent at the target SpO2 (95% CI -301.26 to 116.96). CONCLUSION: FreeO2 technology could significantly enhance the efficiency of the health system by reducing per-patient costs at 180 days. A study with a larger patient sample needs to be carried out to confirm these preliminary results. TRIAL REGISTRATION NUMBER: NCT01393015; Post-results.

Inhibition of Helicobacter pylori Glu-tRNAGln amidotransferase by novel analogues of the putative transamidation intermediate.

Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.

tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP: mechanistic and evolutionary implications.

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of -TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3'-OH oxygen of the 3'-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by chloramphenicol analogs.

Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA(Gln) (Gln-tRNA(Gln)) and/or asparaginyl-tRNA(Asn) (Asn-tRNA(Asn)) are synthesized via an indirect pathway involving side chain amidation of misacylated glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) and/or aspartyl-tRNA(Asn) (Asp-tRNA(Asn)) by an amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active competitive inhibitor of the transamidase activity with respect to Asp-tRNA(Asn) (K(m)=2µM), with a K(i) value of 27µM.

A C-truncated glutamyl-tRNA synthetase specific for tRNA(Glu) is stimulated by its free complementary distal domain: mechanistic and evolutionary implications.

Faithful translation of the genetic code is mainly based on the specificity of tRNA aminoacylation catalyzed by aminoacyl-tRNA synthetases. These enzymes are comprised of a catalytic core and several appended domains. Bacterial glutamyl-tRNA synthetases (GluRS) contain five structural domains, the two distal ones interacting with the anticodon arm of tRNA(Glu). Thermus thermophilus GluRS requires the presence of tRNA(Glu) to bind ATP in the proper site for glutamate activation. In order to test the role of these two distal domains in this mechanism, we characterized the in vitro properties of the C-truncated Escherichia coli GluRSs N(1-313) and N(1-362), containing domains 1-3 and 1-4, respectively, and of their N-truncated complements GluRSs C(314-471) (containing domains 4 and 5) and C(363-471) (free domain 5). These C-truncated GluRSs are soluble, aminoacylate specifically tRNA(Glu), and require the presence of tRNA(Glu) to catalyze the activation of glutamate, as does full-length GluRS(1-471). The k(cat) of tRNA glutamylation catalyzed by N(1-362) is about 2000-fold lower than that catalyzed by the full-length E. coli GluRS(1-471). The addition of free domain 5 (C(363-471)) to N(1-362) strongly stimulates this k(cat) value, indicating that covalent connectivity between N(1-362) and domain 5 is not required for GluRS activity; the hyperbolic relationship between domain 5 concentration and this stimulation indicates that these proteins and tRNA(Glu) form a productive complex with a K(d) of about 100 microM. The K(d) values of tRNA(Glu) interactions with the full-length GluRS and with the truncated GluRSs N(1-362) and free domain 5 are 0.48, 0.11, and about 1.2 microM, respectively; no interaction was detected between these two complementary truncated GluRSs. These results suggest that in the presence of these truncated GluRSs, tRNA(Glu) is positioned for efficient aminoacylation by the two following steps: first, it interacts with GluRS N(1-362) via its acceptor-TPsiC stem loop domain and then with free domain 5 via its anticodon-Dstem-biloop domain, which appeared later during evolution. On the other hand, tRNA glutamylation catalyzed by N(1-313) is not stimulated by its complement C(314-471), revealing the importance of the covalent connectivity between domains 3 and 4 for GluRS aminoacylation activity. The K(m) values of N(1-313) and N(1-362) for each of their substrates are similar to those of full-length GluRS. These C-truncated GluRSs recognize only tRNA(Glu). These results confirm the modular nature of GluRS and support the model of a "recent" fusion of domains 4 and 5 to a proto-GluRS containing the catalytic domain and able to recognize its tRNA substrate(s).

Plasmodial aspartyl-tRNA synthetases and peculiarities in Plasmodium falciparum.

Distinctive features of aspartyl-transfer RNA (tRNA) synthetases (AspRS) from the protozoan Plasmodium genus are described. These apicomplexan AspRSs contain 29-31 amino acid insertions in their anticodon binding domains, a remarkably long N-terminal appendix that varies in size from 110 to 165 amino acids and two potential initiation codons. This article focuses on the atypical functional and structural properties of Plasmodium falciparum cytosolic AspRS, the causative parasite of human malaria. This species encodes a 626 or 577 amino acids AspRS depending on whether initiation starts on the first or second in-frame initiation codon. The longer protein has poor solubility and a propensity to aggregate. Production of the short version was favored as shown by the comparison of the recombinant protein with endogenous AspRS. Comparison of the tRNA aminoacylation activity of wild-type and mutant parasite AspRSs with those of yeast and human AspRSs revealed unique properties. The N-terminal extension contains a motif that provides unexpectedly strong RNA binding to plasmodial AspRS. Furthermore, experiments demonstrated the requirement of the plasmodial insertion for AspRS dimerization and, therefore, tRNA aminoacylation and other putative functions. Implications for the parasite biology are proposed. These data provide a robust background for unraveling the precise functional properties of the parasite AspRS and for developing novel lead compounds against malaria, targeting its idiosyncratic domains.

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. L-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K(i) values in the micromolar range (4-27 microM for human mt- and mammalian cyt-AspRSs). 5'-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.

Mechanism of a GatCAB amidotransferase: aspartyl-tRNA synthetase increases its affinity for Asp-tRNA(Asn) and novel aminoacyl-tRNA analogues are competitive inhibitors.

The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.

Synthesis of beta-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases.

The aminoacyl-beta-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-beta-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-beta-ketophosphonate-adenosine (Gln-KPA), and have tested them as inhibitors of their cognate aaRS, and of a non-cognate aaRS. Glu-KPA is a competitive inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS) with a K(i) of 18microM with respect to its substrate glutamate, and binds at one site on this monomeric enzyme; the non-cognate Gln-KPA also binds this GluRS at one site, but is a much weaker (K(i)=2.9mM) competitive inhibitor. By contrast, Gln-KPA inhibits E. coli glutaminyl-tRNA synthetase (GlnRS) by binding competitively but weakly at two distinct sites on this enzyme (average K(i) of 0.65mM); the non-cognate Glu-KPA shows one-site weak (K(i)=2.8mM) competitive inhibition of GlnRS. These kinetic results indicate that the glutamine and the AMP modules of Gln-KPA, connected by the beta-ketophosphonate linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module. This model suggests that similar structural constraints could affect the binding of Glu-KPA to the active site of mammalian cytoplasmic GluRSs, which are evolutionarily much closer to bacterial GlnRS than to bacterial GluRS. This possibility was confirmed by the fact that Glu-KPA inhibits bovine liver GluRS 145-fold less efficiently than E. coli GluRS by competitive weak binding at two distinct sites (average K(i)=2.6mM). Moreover, these kinetic differences reveal that the active sites of bacterial GluRSs and mammalian cytoplasmic GluRSs have substantial structural differences that could be further exploited for the design of better inhibitors specific for bacterial GluRSs, promising targets for antimicrobial therapy.

Optical ultrawideband monocycle pulse generation based on cross-gain modulation in a semiconductor optical amplifier.

A novel method for generating ultrawideband (UWB) monocycle pulses based on cross-gain modulation (XGM) in a semiconductor optical amplifier (SOA) is proposed and experimentally demonstrated. Thanks to the XGM in the SOA, a pair of polarity-reversed optical Gaussian pulses is generated at the output of the SOA, to which a Gaussian pulse pump and a continuous-wave probe are applied. The two polarity-reversed optical pulses are then time delayed by two cascaded fiber Bragg gratings to introduce a time delay difference. A UWB monocycle pulse with a full width at half-maximum of 48 ps and a fractional bandwidth of 188% is generated at the output of a high-speed photodetector.

A weak Fe-O bond in the oxygenated complex of the nitric-oxide synthase of Staphylococcus aureus.

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.