In vitro measurement of flow rate variability in neonatal IV therapy with and without the use of check valves.

BACKGROUND: In multi-infusion IV therapy, the actual volume delivered to the neonate can vary over time. To reduce flow rate variability, check valves can be used. A check valve allows flow through the valve in only one direction. OBJECTIVE: To evaluate flow rate variability in a low flow dual-infusion setup with and without check valves. METHODS: The effect of changing the height of and adding syringes to the IV-administration set was tested with and without check valves in an in vitro dual-infusion setup with in-line flow meters. The pre-programmed flow rates were 2.5 and 0.1 ml/h. RESULTS: Twenty-four tests of 90 minutes were performed. Time to reach 75% of the pre-programmed 0.1 ml/h flow rate was >20 minutes. The highest total delivered volume during a test was (mean ± SD) 56 ± 8% of the expected delivery for tests without check valves, and diminished to 12 ± 24% of the expected delivery for check valves with a higher opening pressure. CONCLUSIONS: The actual flows and the total delivered volume in low flow dual-infusion setups are less than expected on the pre-programmed flow-rate. These findings emphasize the need for the development of more accurate delivery systems for drugs and fluids in neonatology. Caregivers should be aware of these findings, and optimise the delivery of IV substances by making use of check valves with low opening pressures and by minimising compliance and volume of the IV-administration set. Furthermore, changes in the relative height between pumps and catheter tip should be minimized.

Validation of a dental image analyzer tool to measure alveolar bone loss in periodontitis patients.

BACKGROUND AND OBJECTIVE: Radiographs are an essential adjunct to the clinical examination for periodontal diagnoses. Over the past few years, digital radiographs have become available for use in clinical practice. Therefore, the present study investigated whether measuring alveolar bone loss, using digital radiographs with a newly constructed dental image analyzer tool was comparable to the conventional method, using intra-oral radiographs on film, a light box and a Schei ruler. MATERIAL AND METHODS: Alveolar bone loss of the mesial and distal sites of 60 randomly selected teeth from 12 patients with periodontitis was measured using the conventional method, and then using the dental image analyzer tool, by five dentists. The conventional method scored bone loss in categories of 10% increments relative to the total root length, whereas the software dental image analyzer tool calculated bone loss in 0.1% increments relative to the total root length after crucial landmarks were identified. RESULTS: Both methods showed a high interobserver reliability for bone loss measurements in nonmolar and molar sites (intraclass correlation coefficient > or = 0.88). Also, a high reliability between both methods was demonstrated (intraclass correlation coefficient nonmolar sites, 0.98; intraclass correlation coefficient molar sites, 0.95). In addition, the new dental image analyzer tool showed a high sensitivity (1.00) and a high specificity (0.91) in selecting teeth with > or = 50% or < 50% alveolar bone loss in comparison with the conventional method. CONCLUSION: This study provides evidence that, if digital radiographs are available, the dental image analyzer tool can reliably replace the conventional method for measuring alveolar bone loss in periodontitis patients.

MauE and MauD proteins are essential in methylamine metabolism of Paracoccus denitrificans.

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C1-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the beta-subunit and low levels of the alpha-subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the beta-subunit and that the absence of each of these proteins leads to production of a non-functional beta-subunit which becomes rapidly degraded.

Mutational analysis of mau genes involved in methylamine metabolism in Paracoccus denitrificans.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrificans is under control of a LysR-type transcriptional activator.

Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR-type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.

Identification of a two-component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans.

Upstream of the moxFJGIR genes of Paracoccus denitrificans a regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes, moxZ, moxY and moxX, which are transcribed opposite to moxF and which encode proteins of 16.4, 48.2 and 24.5 kDa, respectively. Computer alignment analysis revealed that the gene products of moxY and moxX have homology with the protein histidine kinases and the response regulators, respectively, forming the two-component regulatory systems. No significant homology of the moxZ gene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified in Escherichia coli in a heterologous expression system. Mutants with an insertion of a kanamycin-resistance marker in moxZ, moxY and moxX were isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In the moxZ mutant both subunits of methanol dehydrogenase and cytochrome c551i were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of a moxZ-lacZ transcriptional fusion was found. Complementation of the mutation was observed after addition of the three genes moxZ, Y and X, in trans. This indicates that the two-component regulatory system is involved in activation of the moxF promoter. A mutant with an unmarked deletion in moxZ was isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of the moxF-lacZ transcriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two-component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenase synthesis.