Replacing risk-based early-onset-disease prevention with intrapartum group B streptococcus PCR testing.

OBJECTIVE: To evaluate the effect of a rapid PCR-based group B streptococcus (GBS) test on length of stay in hospital among newborns, antibiotic use, and GBS-early-onset-disease (EOD) incidence. METHODS: We conducted a before and after service evaluation including term deliveries between 1st January and 12th November 2014 (6688 deliveries). Length of stay in the hospital, GBS-EOD incidence and antibiotic use were evaluated. RESULTS: We recorded three confirmed and 74 possible cases of GBS-EOD in Phase 1, and 85 possible cases in Phase 2. In newborns with suspected infection, the introduction of the rapid test was related to a decreased length of stay on the pediatric care unit by 1.16 days (p = 0.01), and an increase in the length of stay on the mother-and-baby ward by 1.11 days (p < 0.001). No increase in antibiotics was noted. CONCLUSION: The introduction of a point of care test was associated with a reduction in length of stay in the pediatric care unit, without an increase in antibiotic use. This test could improve the accuracy of GBS colonization detection, and help to prevent intrapartum transmission as no verified GBS-EOD cases were recorded with the intrapartum PCR algorithm.

Viral RNA replication in association with cellular membranes.

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a "tip-anchored" protein attached to the membrane by an amphipathic alpha-helix (NS5A) and a "tail-anchored" posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic alpha-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

Virus-specific capping of tobacco mosaic virus RNA: methylation of GTP prior to formation of covalent complex p126-m7GMP.

In capping cellular mRNAs, a covalent GMP-enzyme intermediate leads to formation of G(5')ppp(5')N at the 5' end of the RNA, which is modified by methylation catalyzed by guanine-7-methyltransferase. Here we show that isolated membranes from tobacco mosaic virus (TMV)-infected plant or insect cells expressing TMV replicase protein p126, synthesized m7GTP using S-adenosylmethionine (AdoMet) as the methyl donor, and catalyzed the formation of a covalent guanylate-p126 complex in the presence of AdoMet. The methyl group from AdoMet was incorporated into p126, suggesting that the complex consisted of m7GMP-p126. Thus, TMV and alphaviruses, despite their evolutionary distance, share the same virus-specific capping mechanism.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.

Non-invasive measurement of cardiac output: whole-body impedance cardiography in simultaneous comparison with thermodilution and direct oxygen Fick methods.

OBJECTIVE: To determine the reliability of whole-body impedance cardiography (ICGWB), with electrodes attached to wrists and ankles, in the measurement of cardiac output (CO) on the basis of simultaneous comparison with thermodilution (TD) and direct oxygen Fick (Fick) methods. DESIGN: Prospective clinical study. SETTING: A surgical intensive care unit at a university hospital. PATIENTS: Thirty consecutive subjects undergoing a coronary artery bypass surgery were investigated preoperatively. MEASUREMENTS: ICGWB derived CO was measured simultaneously with the TD and Fick methods to establish the biases and limits of agreement (LA) between the methods. RESULTS: The results obtained by ICGWB and the invasive methods showed good agreement. The bias and LA between COTD and COICG were 0.00 l/min: 1.37 and 1.37 l/min, respectively, and were close to those obtained between COTD and COFICK, 0.32 l/min; 1.74 and -1.10 l/min. The bias and LA between the COFICK and COICG were -0.32 l/min; -2.24 and 1.60 l/min respectively. The repeatability value of consecutive single measurements for ICGWB (RVICG = 0.57 l/min) was much better than for the TD method (RVTD = 1.10 l/min). CONCLUSION: There was close agreement between the results of the three methods in the measurement of CO. In sedated preoperative patients the accuracy of ICGWB is within clinically acceptable limits and its repeatability is excellent. ICGWB provides a useful alternative to the TD and Fick methods in cases where the pressures supplied by the pulmonary artery catheter are not essential.

An improved biotonometric method for determination of haemoglobin oxygen equilibrium curves.

A new biotonometric method for the determination of haemoglobin oxygen equilibrium curves is described. The procedure is based on the mixing of an oxygen-consuming organism, the yeast cell (Saccharomyces cerevisiae), with an oxidized blood/plasma mixture. The yeast cell consumes oxygen at a uniform rate, thus reducing the partial pressure of oxygen in the mixture. This in turn induces the dissociation of oxygen in a characteristic manner. The study of the whole blood samples from 26 healthy volunteer subjects gave the following results: p50 = 3.63 kPa +/- 0.23 kPa (mean +/- 1 S.D.); Hill slope n = 2.44 +/- 0.16; and CO2 Bohr factor = -0.47 +/- 0.11. For the within-run imprecision the coefficients of variation for the different parameters were: p50 C.V. = 4.4%; Hill slope n C.V. = 4.7%; and CO2 Bohr factor C.V. = 19%. The determination can be carried out with simple equipment: a blood gas analyzer with a coupled recorder.

Coenzyme Q10 supplementation and recovery from ischemia in senescent rat myocardium.

Many studies have suggested that parenteral administration of coenzyme Q10 (Q10) protects the myocardium of young experimental animals from post-ischemic reperfusion injury. Although parenteral administration, in contrast to per os supplementation, seems to elevate coenzyme Q concentrations in heart tissue, it is not suitable for prophylactic use. In addition, the incidence of ischemic events is greatest in older age. We studied the effect of Q10 supplementation on myocardial postischemic recovery in 18-month-old Wistar rats. The treated group (n=9) received 10 mg/kg/day of Q10 for 8 weeks in their chow while the normal chow of the control group (n=9) contained less than 0.5 mg/kg/day of Q10. The treatment clearly elevated plasma Q10 concentration (286 +/- 25 micromol/l and 48 +/- 30 micromol/l, treated and controls, respectively, p<0.0001) but neither Q9 nor Q10 concentrations in heart tissue were affected by the supplementation. The isolated perfused hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The preischemic values of developed pressure (DP) but not contractility (+DP/delta t) and relaxation (-DP/delta t) were improved by Q10 supplementation (p=0.034, p=0.057 and p=0.13, respectively) while in postischemic recovery no differences were observed between the groups (p>0.05 at all time points). Also, in myocardial flow, myocardial oxygen consumption (MVO2) and myocardial aerobic efficiency (DP/MVO2) the groups did not differ at any time points. Although dietary Q10 supplementation clearly elevated plasma Q10 concentrations in senescent rats, the coenzyme Q contents in heart tissue and myocardial recovery from ischemia were not affected. However, it is possible that the site of action for the reported beneficial effects of Q10 is in the coronary endothelium rather than myocardium itself.

Return of the question "why": advantages of exploring pre-existing explanations.

We suggest that there are many advantages in thoroughly exploring the causal explanations given by clients or members of their social network to account for their problems. Specific interviewing techniques are presented to uncover clients' causal explanations or their impressions about the causal explanations of others. Various advantages of exploring these explanations are discussed. They include improved cooperation, development of "systemic empathy," detachment from the explanations of other professionals, recognition and avoidance of coalitions, loosening of firmly held explanations, dilution of noxious explanations, generation of new and positive explanations toward solutions, and taking a bird's-eye view or meta position about such explanations. We conclude that acceptance and appreciation of the human tendency to believe in causal explanations is a fruitful way to enhance interaction between clinicians and clients.

Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.

After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

The effects of palmitoylation on membrane association of Semliki forest virus RNA capping enzyme.

The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes. To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue. Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys418, Cys419, and Cys420 resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mM sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein. Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.

Sialic acid secretion of patients with gastric and duodenal ulcers in the dose-response pentastrin test.

The secretion of HCl and sialic acid into the gastric juice was investigated in 13 patients with gastric, 52 with duodenal ulcers and 27 control patients, during the dose-response pentagastrin test. The basal output of sialic acid was clearly highest in patients with gastric ulcers, and the differences between these and other groups of patients were significant. During pentagastrin stimulation a significant initial decrease was found in patients with gastric ulcers with small doses, and the minimum was reached with a dose of 0.1 micrograms/kg/h. After increasing the dose, an increase in sialic acid output occurred in all groups, reaching its maximum with a pentagastrin infusion rate of 1.0 micrograms/kg/h. The basal concentration of sialic acid was also highest in patients with gastric ulcers. In all groups the concentration decreased during the stimulation, which obviously depended on the sialic output not increasing in the same relationship as the volume of gastric secretion.

Thiol metabolism in preterm infants during the first week of life.

BACKGROUND: Oxidative stress is implicated in the pathogenesis of several complications of prematurity. The glutathione cycle is one of the most important intracellular antioxidant systems. The synthesis of glutathione may not be adequate in preterm neonates because of the low levels of cysteine available. The aim of this study was to evaluate cysteine and glutathione metabolism during the first week of life in preterm infants. METHODS: Plasma and erythrocyte thiol concentrations were measured in 78 preterm infants with a birthweight of 500-1500 g, and erythrocyte glutamate-cysteine ligase (GCL), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferases (GST) and glucose 6-phosphatedehydrogenase (G6PDH) in 26 infants with a birthweight of 1000-1500 g. RESULTS: The mean (SD) plasma glutathione concentration increased from day 0 to day 1 (14.9 (7.1) vs. 27.7 (11.9) micromol/L, p < 0.001), and then decreased. The plasma cysteine concentration changed in the opposite direction (172 (59) vs. 129 (42) micromol/L, p < 0.01). In infants with respiratory distress syndrome (RDS) the mean plasma glutathione concentration, but not cysteine, was lower on day 0 compared with infants without RDS (11.7 (5.2) vs. 21.4 (5.6) micromol/L, p < 0.01). Erythrocyte glutathione concentration decreased during the first week of life, whereas erythrocyte cysteine concentration increased significantly from day 3 to day 7 (p < 0.01). Erythrocyte cysteine and glutathione concentrations had a positive correlation. The GCL and GR activities did not change, but GST and G6PDH activities decreased during the first week (p < 0.01). GPx activity decreased until day 3 (p < 0.01) and was higher on day 0 and day 1 in infants with RDS. CONCLUSIONS: Very low birthweight infants have an initial increase in plasma glutathione and initial decrease in plasma cysteine level during the first week of life, and also a positive correlation between erythrocyte cysteine and glutathione levels.

Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities.

The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covalent complex m7GMP-nsP1. These reactions are virus specific and thus potential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the large alphavirus-like superfamily. Mutations of D64, D90, R93, C135, C142, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1. All MT-negative mutants lost also the GT activity, confirming that methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT reaction without destroying MT activity. Conservation of residues essential for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Residues D64 and D90 were necessary for AdoMet binding, as measured by UV cross-linking. Secondary structure predictions of nsP1 and other proteins of the superfamily place these residues in positions corresponding to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related.

Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein.

A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4. All nsPs were membrane associated, and nsP2 was also transported to the nucleus. The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1.

Bacterial overgrowth, intestinal transit, and nutrition after total gastrectomy. Comparison of a jejunal pouch with Roux-en-Y reconstruction in a prospective random study.

BACKGROUND: Jejunal pouches after total gastrectomy have been introduced to diminish postgastrectomy symptoms and improve nutrition. However, the effect of a pouch on the intestinal bacteriology and transit is controversial. METHODS: Bacterial overgrowth was measured with the glucose breath test and the mouth-to-caecum transit time (MCT) by means of the lactulose breath test after total gastrectomy and Roux-en-Y reconstruction in 24 patients with a pouch (Pouch group) and in 22 patients without a pouch (Roux-en-Y group). Postoperative symptoms were evaluated with a standard questionnaire, and nutrition was measured by blood chemistry and weight loss. RESULTS: MCTT was 110 +/- 44 min in the Roux-en-Y group and 117 +/- 44 min in the Pouch group (NS). Eighty-six per cent of the patients in the Roux-en-Y group and 91% of the patients in the Pouch group had bacterial overgrowth (NS). Transit time was shorter in patients with severe dumping than patients without dumping (60 +/- 28 min versus 115 +/- 41 min; P = 0.04). Maximal hydrogen concentration in the glucose breath test correlated negatively with serum albumin and iron concentrations and with postoperative weight loss, and positively with serum alkaline phosphatase activity. CONCLUSIONS: Bacterial overgrowth is common in the upper intestine after total gastrectomy. Pouch reconstruction does not delay the transit of liquids. Bacterial overgrowth may be one of the main aetiologic factors in postgastrectomy malnutrition.

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.