Benchmarking the use of blood products in cardiac surgery to stimulate awareness of transfusion behaviour

Introduction

Cardiac operations account for a large proportion of the blood transfusions given each year, leading to high costs and an increased risk to patient safety. Therefore, it is important to explore initiatives to reduce transfusion rates. This study aims to provide a benchmark for transfusion practice by inter-hospital comparison of transfusion rates, blood product use and costs related to patients undergoing coronary artery bypass grafting (CABG), valve surgery or combined CABG and valve surgery.

Methods

Between 2010 and 2013, patients from four Dutch hospitals undergoing CABG, valve surgery or combined CABG and valve surgery (n = 11,150) were included by means of a retrospective longitudinal study design.

Results

In CABG surgery the transfusion rate ranged between 43 and 54%, in valve surgery between 54 and 67%, and in combined CABG and valve surgery between 80 and 88%. With the exception of one hospital, the trend in transfusion rate showed a significant decrease over time for all procedures. Hospitals differed significantly in the units of blood products given to each patient, and in the use of specific transfused combinations of blood products, such as red blood cells (RBCs) and a combination of RBCs, fresh frozen plasma (FFP) and platelets.

Conclusion

This study indicates that benchmarking blood product usage stimulates awareness of transfusion behaviour, which may lead to better patient safety and lower costs. Further studies are warranted to improve awareness of transfusion behaviour and increase the standardisation of transfusion practice in cardiac surgery.

     

The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.     

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.     

The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn²⁺ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn²⁺ oxidation and/or secretion of the Mn²⁺-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn²⁺ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn²⁺-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn²⁺ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.In a number of studies during the last three decades it has been shown that various microbial species are able to stimulate the oxidation of Mn²⁺ through direct catalysis. These organisms produce proteinaceous macromolecules which catalyze the oxidation reaction. Manganese oxidations by a soil Arthrobacter species (24), Oceanospirillum and Vibrio strains (2, 3), Pseudomonas putida MnB1 (22, 30), Leptothrix discophora SS-1 (1, 11), and marine Bacillus strain SG-1 (23) are examples in which enzymes are most likely involved in the process. P. putida MnB1 produces a soluble protein which catalytically oxidizes Mn²⁺ in cell extracts (22). Manganese-oxidizing proteins from L. discophora SS-1 (1, 11) and from the spore coats of Bacillus strain SG-1 (43) have been identified on polyacrylamide gels. The oxidizing proteins have not been quantitatively purified or analyzed so far. In Bacillus strain SG-1, an operon containing seven genes appears to be involved in Mn²⁺ oxidation (46). One of these genes encodes a 137-kDa protein related to the family of multicopper oxidases (47). In a previous study we reported the isolation of a structural gene and its promoter postulated to be involved in Mn²⁺ oxidation in L. discophora (19). The encoded protein also contains the copper-binding signatures of multicopper oxidases. The oxidase-related proteins may represent Mn²⁺-oxidizing enzymes (44), but evidence supporting this hypothesis is still lacking.In this paper we describe a genetic analysis of Mn²⁺ oxidation in a freshwater Pseudomonas strain, strain GB-1. In a previous study (32) this strain was preliminarily identified as a Pseudomonas fluorescens strain, but more recent data (see Materials and Methods) indicate that it should be identified as a P. putida strain. When supplied with Mn²⁺ ions, the cells deposit manganese oxide around the outer membrane in the early stationary growth phase (32). They form brown colonies on Mn²⁺-containing agar. Experiments performed with cell extracts indicated that Mn²⁺ oxidation is catalyzed by a protein. The Mn²⁺-oxidizing factor was partially purified, and electrophoresis on an acrylamide gradient gel revealed oxidizing proteins with apparent molecular weights of ca. 250,000 and 180,000 (32). An additional oxidizing factor with a lower molecular weight (ca. 130,000) was identified in another study by using different isolation and electrophoretic procedures (16). It has been suggested that the Mn²⁺-oxidizing protein isolated is part of a larger complex which disintegrates into smaller fragments that retain activity (32). The protein is supposed to be located in the outer membrane of the bacteria. It has not been chemically characterized, and nothing is known about its cellular function or about the possible involvement of other cellular components, such as electron carriers, in Mn²⁺ oxidation.We used transposon mutagenesis to identify genes relevant to the Mn²⁺-oxidizing process in P. putida GB-1. One of these genes appeared to be part of the cytochrome c maturation operon. Transposon insertion in this gene not only abolished Mn²⁺ oxidation but also led to secretion of siderophores and porphyrins.An accompanying report on the involvement of the cytochrome c maturation operon in Mn²⁺ oxidation in P. putida MnB1 (14) supports our findings.     

Schistosoma mansoni does not and cannot oxidise fatty acids,but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [¹⁴C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no ¹⁴CO₂ production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.     

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate–type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.     

cumA, a Gene Encoding a Multicopper Oxidase, Is Involved in Mn2+ Oxidation in Pseudomonas putida GB-1

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn²⁺. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu²⁺ increased the Mn²⁺-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu²⁺. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn²⁺-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn²⁺ and that CumB is required for optimal growth of P. putida GB-1-002.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.     

The acoustic climate of plant communities

Summary During summer season and winter season of 1971 and 1972, the acoustic properties, defined as the acoustic climate, of 8 semi-natural plant communities in the Netherlands were investigated. It appeared that each plant community has its own acoustic climate as a consequence of the specific species composition and the specific acoustic properties of the soil. Seasonal changes influenced the acoustic climate to some extent, since during summer there is a better attenuation of low frequencies and in winter of mid frequencies. Comparing all plant communities the total variation in attenuation differed from 29.6 dB/100 m at 1250 Hz (cps) in winter to 74.6 dB/100 m at 10,000 Hz in summer, as is shown in Table 3. In general best attenuation occurred at low and high frequencies in both seasons, which has to be attributed to soil and foliage characteristics.