Implementation of the 2015 European Society of Cardiology guidelines for the management of infective endocarditis in the Netherlands

Because the occurrence of infective endocarditis (IE) continues to be associated with high mortality, a working group was created by the Dutch Society of Cardiology to examine how the most recent European Society of Cardiology (ESC) guidelines for IE management could be implemented most effectively in the Netherlands. In order to investigate current Dutch IE practices, the working group conducted a country-wide survey. Based on the results obtained, it was concluded that most ESC recommendations could be endorsed, albeit with some adjustments. For instance, the suggested pre-operative screening and treatment of nasal carriers of Staphylococcus aureus as formulated in the ESC guideline was found to be dissimilar to current Dutch practice, and was therefore made less restrictive. The recently adapted ESC diagnostic criteria for IE were endorsed, while the practical employment of the relevant diagnostic techniques was simplified in an adapted flowchart. In addition, the presence of a multidisciplinary, so-called ‘endocarditis team’ in tertiary centres was proposed as a quality indicator. An adapted flowchart specifically tailored to Dutch practice for microbiological diagnostic purposes was constructed. Lastly, the working group recommended the Stichting Werkgroep Antibioticabeleid (SWAB; Dutch Working Party on Antibiotic Policy) guidelines for IE treatment instead of the antibiotic regimens proposed by the ESC.

     

Short-term and long-term effects of tannins on nitrogen mineralisation and litter decomposition in kauri (Agathis australis (D. Don) Lindl.) forests

Kauri (Agathis australis (D. Don) Lindl.) occurs naturally in the warm temperate forest of northern New Zealand where it grows mixed with angiosperm tree species. Below mature kauri trees thick organic layers develop in which large amounts of nitrogen are accumulated. This nitrogen seems to be inaccessible to plants. While litter quality can explain the low decomposition rate below kauri, it is not known what causes the accumulation of nitrogen. We hypothesised that kauri tannins reduce nitrogen mineralisation and litter decomposition below kauri. We further hypothesised that high tannin concentrations in the soil would increase the availability of dissolved organic nitrogen relative to the availability of inorganic nitrogen. To test these hypotheses a laboratory incubation was carried out for 1 year. Purified tannins of kauri and of two other common New Zealand tree species were added to samples of the soil organic layer from under a kauri tree. The results suggest that during the first month of incubation the added tannins reduced nitrogen availability by sequestering proteins or by stimulating nitrogen immobilisation. In the long-term, the reduced nitrogen release, which was found following tannin addition, seems attributable to the complexation of proteins by tannins. It further appeared that the addition of tannins did not change the ratio of dissolved organic nitrogen to inorganic nitrogen in the long-term. We conclude that the effect of kauri tannins on nitrogen release offers a good explanation for the accumulation of nitrogen below kauri trees.     

End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4

DNA double-strand break repair by non-homologous end-joining (NHEJ) is generally considered to be an imprecise repair pathway. In order to study repair of a blunt, 5' phosphorylated break in the DNA of mammalian fibroblasts, we used the E. coli cut-and-paste type transposon Tn5. We found that the Tn5 transposase can mediate transposon excision in Chinese hamster cell lines. Interestingly, a blunt 5' phosphorylated break could efficiently be repaired without loss of nucleotides in wild type fibroblasts. Catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) deficiency reduced the efficiency of joining four-fold without reducing precision, whereas both efficiency and accuracy of joining were affected in Ku80 or XRCC4 mutant cell lines. These results show that both the DNA-PK and the XRCC4/ligase IV complexes are required for NHEJ and that other, more error-prone, repair processes cannot efficiently substitute for joining of blunt breaks produced in living cells. Interestingly, the severity of the end-joining defect differs between the various mutants, which may explain the difference in the severity of the phenotypes, which have been observed in the corresponding mouse models.     

The role of DNA dependent protein kinase in synapsis of DNA ends

DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK_CS) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK_CS, which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.     

A trait‐based approach reveals the feeding selectivity of a small endangered Mediterranean fish

Functional traits are growing in popularity in modern ecology, but feeding studies remain primarily rooted in a taxonomic‐based perspective. However, consumers do not have any reason to select their prey using a taxonomic criterion, and prey assemblages are variable in space and time, which makes taxon‐based studies assemblage‐specific. To illustrate the benefits of the trait‐based approach to assessing food choice, we studied the feeding ecology of the endangered freshwater fish Barbus meridionalis. We hypothesized that B. meridionalis is a selective predator which food choice depends on several prey morphological and behavioral traits, and thus, its top‐down pressure may lead to changes in the functional composition of in‐stream macroinvertebrate communities. Feeding selectivity was inferred by comparing taxonomic and functional composition (13 traits) between ingested and free‐living potential prey using the Jacob's electivity index. Our results showed that the fish diet was influenced by 10 of the 13 traits tested. Barbus meridionalis preferred prey with a potential size of 5–10 mm, with a medium–high drift tendency, and that drift during daylight. Potential prey with no body flexibility, conical shape, concealment traits (presence of nets and/or cases, or patterned coloration), and high aggregation tendency had a low predation risk. Similarly, surface swimmers and interstitial taxa were low vulnerable to predation. Feeding selectivity altered the functional composition of the macroinvertebrate communities. Fish absence favored taxa with weak aggregation tendency, weak flexibility, and a relatively large size (10–20 mm of potential size). Besides, predatory invertebrates may increase in fish absence. In conclusion, our study shows that the incorporation of the trait‐based approach in diet studies is a promising avenue to improve our mechanistic understanding of predator–prey interactions and to help predict the ecological outcomes of predator invasions and extinctions.     

Low soil water and nutrient availability below New Zealand kauri (<Emphasis Type="Italic">Agathis australis</Emphasis> (D. Don) Lindl.) trees increase the relative fitness of kauri seedlings

Tree species can affect the soil they are growing on and this might influence their fitness. The New Zealand gymnosperm tree species kauri (Agathis australis (D. Don) Lindl.) which grows in mixed angiosperm–gymnosperm forests has a substantial effect upon the soil. We studied the hypotheses that: (1) low soil moisture availability below mature kauri trees hampers growth of kauri seedlings and angiosperm seedlings, (2) low nutrient availability below kauri trees hampers only angiosperm seedlings, and (3) angiosperm seedlings are hampered more than kauri seedlings by the conditions below kauri trees. We tested these hypotheses by planting seedlings of kauri and mapau (Myrsine australis (A. Rich) Allan) under kauri trees and applying the following treatments: removal of herbs, removal of litter, removal of nutrient limitation, and elimination of root competition of mature kauri trees. The results indicate that low soil moisture availability, or the combination of low soil moisture availability and low nutrient fertility, hampers the growth of kauri as well as mapau seedlings below kauri trees. The mapau seedlings are hampered relatively more than the kauri seedlings which might result in an increased relative fitness of the latter.     

The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK_CS) and the XRCC4/ligase IV complex. Activation of the DNA-PK_CS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK_CS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK_CS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK_CS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.DNA double-strand breaks (DSBs) classify among the most detrimental DNA damages, because they have the ability to cause chromosome breakage and translocations. DSBs are readily caused by common exogenous and endogenous agents, including certain oxygen radicals, products of normal metabolism, and ionizing radiation. Effective genomic maintenance therefore requires the presence of a mechanism to repair DSBs. DSB repair in eukaryotic cells is executed by either homologous recombination or by nonhomologous end joining (NHEJ) (15, 30).In vertebrates, DSB repair is not only essential for genomic maintenance, but also for the development of a working immune system. The assembly of immunoglobulin or T-cell receptor genes via V(D)J recombination routinely necessitates the introduction and subsequent NHEJ-mediated repair of DSBs (13).The NHEJ pathway facilitates DSB repair by direct ligation of the two ends of a broken DNA molecule (31, 36). This requires the sequential loading of several enzymes on both DNA ends. The first event in NHEJ-mediated repair is the association of a Ku70-Ku80 heterodimer (Ku70/80) with each DNA terminus. The Ku70/80 molecule has a ring-shaped structure, made up by the amino-terminal and central domains of both the Ku70 and the Ku80 polypeptides, which exactly fits a DNA helix in its center (33).The DNA-Ku complex functions as a scaffold to attract the other known NHEJ factors to the DSB. One of the enzymes that are recruited to the DNA-Ku scaffold is the DNA-dependent protein kinase catalytic subunit (DNA-PK_CS), a 469-kDa serine/threonine kinase. The Ku-DNA-PK_CS complex is commonly referred to as DNA-PK. It has been well established that the DNA-PK_CS kinase activity is essential for efficient DSB repair, although the mechanism via which DNA-PK_CS exerts its function is a matter of current debate (19, 35, 36). Several autophosphorylation sites have been mapped in the DNA-PK_CS protein. The most important clusters are found between residues 2609 and 2647 (ABCDE cluster) and between residues 2023 and 2056 (PQR cluster). Phosphorylation of the ABCDE cluster was found to specifically stimulate processing and joining of DNA ends, while PQR phosphorylation reduced the level of DNA end processing (35). These findings prompted a model in which DNA-PK_CS functions as a gatekeeper molecule that regulates access to the DNA termini by changing its phosphorylation status (35). Therefore, DNA-PK_CS autophosphorylation may regulate the next steps in the NHEJ process.These next steps include the processing and joining of DNA ends. Processing enzymes prepare nonligatable DNA termini, primarily blocked ends and incompatible single-strand overhangs, for subsequent ligation by the XRCC4/ligase IV complex. The chemistry of the ligation reaction necessitates the addition of 5′ phosphate groups or the removal of 3′ phosphate groups by polynucleotide kinase (3). Processing of single-strand overhangs is performed by either filling or resection and therefore requires a polymerase or a nuclease, respectively (16, 36). Several enzymes with single-strand filling capability, including polymerase λ, polymerase μ, and terminal deoxynucleotidyltransferase, have been suggested to function as processing enzymes during NHEJ (16). In contrast, only one nuclease has been conclusively shown to play a role in NHEJ: the endonuclease Artemis.Artemis was first described as an essential contributor to V(D)J recombination, catalyzing the opening of hairpin structures at coding ends (17, 21, 24). However, because Artemis deficiency not only causes impairment of V(D)J recombination but also increased sensitivity to DSB-inducing ionizing radiation, it was soon recognized that Artemis may act as a processing enzyme for other types of DNA ends during NHEJ as well. The Artemis protein forms a complex with DNA-PK and carries the endonuclease activity that is necessary for the hairpin opening or overhang processing (14, 17). It is likely that the Artemis protein is recruited to the repair complex by interaction with the DNA-Ku-DNA-PK_CS complex.Because the NHEJ core factors DNA-PK_CS, XRCC4/ligase IV, and Artemis are attracted to a DSB by the DNA-Ku scaffold, we set out to examine the influence of specific deletions of the Ku80 protein on the recruitment and activation of these core factors. It has been previously reported that the Ku80 carboxy terminus is important for effective NHEJ, evidenced by the fact that deletion of the Ku80 carboxy terminus results in markedly increased sensitivity to ionizing radiation and decreased retention of DNA-PK_CS at DNA ends (11). Several authors have suggested that the Ku80 carboxy terminus mediates activation of the DNA-PK_CS kinase and may therefore be directly responsible for regulation of the NHEJ process (11, 12, 25).In contrast to that hypothesis, we here show that the Ku80 carboxy terminus is not an essential prerequisite for recruitment or activation of the DNA-PK_CS kinase in vivo. Surprisingly, however, deletion of the Ku80 carboxy terminus resulted in less efficient phosphorylation of specific DNA-PK_CS autophosphorylation sites and diminished Artemis endonuclease activity. These findings provide a comprehensive explanation for the increased radiation sensitivity that is associated with deletion of the Ku80 carboxy terminus.     

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.