Preoperative systemic inflammation and perioperative myocardial injury: prospective observational multicentre cohort study of patients undergoing non-cardiac surgery.

BACKGROUND: Systemic inflammation is pivotal in the pathogenesis of cardiovascular disease. As inflammation can directly cause cardiomyocyte injury, we hypothesised that established systemic inflammation, as reflected by elevated preoperative neutrophil-lymphocyte ratio (NLR) >4, predisposes patients to perioperative myocardial injury. METHODS: We prospectively recruited 1652 patients aged ≥45 yr who underwent non-cardiac surgery in two UK centres. Serum high sensitivity troponin T (hsTnT) concentrations were measured on the first three postoperative days. Clinicians and investigators were blinded to the troponin results. The primary outcome was perioperative myocardial injury, defined as hsTnT≥14 ng L-1 within 3 days after surgery. We assessed whether myocardial injury was associated with preoperative NLR>4, activated reactive oxygen species (ROS) generation in circulating monocytes, or both. Multivariable logistic regression analysis explored associations between age, sex, NLR, Revised Cardiac Risk Index, individual leukocyte subsets, and myocardial injury. Flow cytometric quantification of ROS was done in 21 patients. Data are presented as n (%) or odds ratio (OR) with 95% confidence intervals. RESULTS: Preoperative NLR>4 was present in 239/1652 (14.5%) patients. Myocardial injury occurred in 405/1652 (24.5%) patients and was more common in patients with preoperative NLR>4 [OR: 2.56 (1.92-3.41); P<0.0001]. Myocardial injury was independently associated with lower absolute preoperative lymphocyte count [OR 1.80 (1.50-2.17); P<0.0001] and higher absolute preoperative monocyte count [OR 1.93 (1.12-3.30); P=0.017]. Monocyte ROS generation correlated with NLR (r=0.47; P=0.03). CONCLUSIONS: Preoperative NLR>4 is associated with perioperative myocardial injury, independent of conventional risk factors. Systemic inflammation may contribute to the development of perioperative myocardial injury. CLINICAL TRIAL REGISTRATION: NCT01842568.

Wingless signaling leads to an asymmetric response to decapentaplegic-dependent signaling during sense organ patterning on the notum of Drosophila melanogaster.

Wnt and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila melanogaster. The Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homologue of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed "polarity" with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, which we suggest is Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells which also receive the Wingless signal. Activation of Hedgehog signaling generates a long-range signal which can promote macrochaete formation in the Wingless activity domain. This signal depends upon decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Our results suggest a novel patterning mechanism which determines sense organ positioning in Drosophila.

How is developmental stability sustained in the face of genetic variation?

The number and arrangement of scutellar bristles on the thorax of Drosophila melanogaster is largely invariant in wild-type stocks. This character therefore appears to be buffered against changes in phenotype, and has previously been described as a canalized character. Mutations that do alter this phenotype increase the variability in bristle number and can reveal otherwise cryptic genetic differences at other loci. This phenomenon is examined and possible mechanisms contributing to stability of this developmental event are discussed, but the notion that the character is canalized is found not to be heuristic.

Genetic relationships between the mutations spade and Sternopleural and the wingless gene in Drosophila development.

In Drosophila melanogaster, there are cases in which gene products contributing to the same developmental event may derive from closely adjacent transcription units and may even share cis-regulatory sequences. Correct recognition of such genomic organization is central to an understanding of developmental mechanisms. The adult phenotypes of combinations between the mutations spade, Sternopleural, and wingless suggest that they are lesions in functionally related genes within the same chromosomal region. wingless mutations fail to complement the recessive mutation spade. The spade mutation, as previously shown, behaves as a lesion in a regulatory site of wingless, sited 5' to the transcription unit, and is concerned with particular postembryonic functions of wingless. While showing wingless-like phenotypes in combination with Sternopleural, even lethal alleles of wingless complement the recessive lethality of Sternopleural alleles. Mutations in Sternopleural increase the severity of wingless phenotypes in many wingless-dependent processes during postembryonic development, and this interaction can occur when the only functional copies of Sp or wg are located in either opposing chromosomes or the same chromosome. This is inconsistent with previous attempts to define Sp as a regulatory allele of wg and explain the phenotypes that result from combinations of Sp and wg by means of transvection. We have analyzed a new EMS-induced allele of Sternopleural that is more severe than the original allele, which also argues for Sp being a separate, mutable genetic locus rather than a regulatory allele of wg. Finally, we have a revertant of Sternopleural (Sp[Rv1]) that behaves as a genetic null allele of wg, but causes ventral-to-dorsal transformations in combination with wg(P), which is not observed in combinations of wg null alleles with wg(P). Because wg(P) is the result of an inversion and because inversions inhibit transvection, the increased severity observed in Sp(Rv1)/wg(P) in comparison to wg(null)/Sp(Rv1) animals cannot be explained by an absence of transvection. Therefore, the two Sternopleural mutations most reasonably define an independent gene located 3' to the wingless gene and having strong functional synergism with it.

Properties of xanthine dehydrogenase variants from rosy mutant strains of Drosophila melanogaster and their relevance to the enzyme's structure and mechanism.

Xanthine dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant files. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]xanthine dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme. [E89K]xanthine dehydrogenase. These both had ultraviolet-visible absorption spectra similar to milk xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and [S357F]xanthine dehydrogenase mutated in the flavin domain, and [G1011E]xanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]xanthine dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD(+)-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the files, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romäo, M. J., Archer, M., Moura, I., Moura. J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270. 1170-1176) from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]xanthine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into xanthine dehydrogenase and in relation to other molybdenum enzymes are discussed.

wingless expression mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development.

We have used conditional wingless genotypes to dissect the role of this gene in late stages of wing disc development. One of these genotypes (wgIL/wg-lacZ) is simultaneously a reporter of wingless transcription and temperature-sensitive for wingless function, and has allowed us to define its pattern of transcription in the absence of wingless activity. The primordia of a subset of the bristles of the notum, which develop in or immediately adjacent to wingless-expressing cells, depend upon wingless activity. The time-course of this contribution and the effect on proneural gene expression together suggest that wingless may regulate the activity of products of the achaete-scute complex in proneural clusters. wingless activity is also required at the presumptive wing margin and is a necessary precondition for the change in proliferation pattern in this region. The involvement of wingless in transducing or mediating positional signals for spatial patterning in imaginal disc development is discussed.

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Establishment of imaginal discs and histoblast nests in Drosophila.

In Drosophila the homeotic genes of the bithorax-complex (BX-C) and Antennapedia-complex (ANT-C) specify the identity of segments. Adult segment primordia are established in the embryo as the histoblast nests of the abdomen and the imaginal discs of the head, thorax and terminalia. We have used a molecular probe for the limb primordia and in vivo culture to describe the nature of the adult primordia in mutants in which the pattern of homeotic gene expression was altered. The results suggest that the histoblast or disc 'mode' of development is initiated by the extended germ band stage through activity of the BX-C and ANT-C and is relatively inflexible thereafter [corrected].

The Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs.

We demonstrate the role of the segment polarity gene patched (ptc) in patterning in the cuticle of the adult fly. Genetic mosaics of a lethal allele of patched show that the contribution of patched varies in a position-specific manner, defining three regions in the wing where ptc clones, respectively, behave as wild-type cells, affect vein formation, or are rarely recovered. Analysis of twin clones demonstrates that the reduced clone frequency results from a proliferation failure or cell loss. In the region where clones upset venation, they autonomously fail to form veins and also non-autonomously induce ectopic veins in adjacent wild-type cells. In heteroallelic combinations with lethal alleles, two viable alleles produce distinct phenotypes: (1) loss of structures and mirror-image duplications in the region where patched clones fail to proliferate; (2) vein abnormalities in the anterior compartment. We propose that these differences reflect independently mutable functions within the gene. We show the pattern of patched transcription in the developing imaginal wing disc in relation to the expression of certain other reporter genes using a novel double-labelling method combining non-radioactive detection of in situ hybridization with beta-galactosidase detection. The patched transcript is present throughout the anterior compartment, with a stripe of maximal intensity along the A/P compartment border extending into the posterior compartment. We propose that the patched product is a component of a cell-to-cell position-signalling mechanism, a proposal consistent with the predicted structure of the patched protein.

Pattern formation in imaginal discs.

Imaginal discs are sacs of folded epithelium arising during embryogenesis. They proliferate during the larval instars, and at metamorphosis secrete the adult cuticle, therefore being responsible for the characteristic surface patterning of this insect. Each disc has intrinsic growth controls and their cell lineage shows constraints known as compartments. Spatial patterns emerge through interaction between adjacent cells. Molecular genetic analysis of mutants with changed pattern has implicated transcription factors, secreted, membrane-bound and growth factor related proteins in the position-signalling mechanism. Their accessibility to contemporary cell biological techniques makes imaginal discs a model system for investigating patterning in animal tissues.

A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched.

The patterning of cells in insect segments requires the exchange of information between cells, which in Drosophila depends on the activity of members of the segment-polarity class of genes. Here we report the molecular characterization of one such gene, patched. We find that patched encodes a large protein with several possible membrane-spanning domains and is expressed in a complex pattern during embryogenesis.

Imaginal discs can be recovered from cultured embryos mutant for the segment-polarity genes engrailed, naked and patched but not from wingless.

Drosophila embryos homozygous for strong mutations in each of the segment-polarity genes wingless (wg), engrailed (en), naked (nkd) and patched (ptc) form a larval cuticle in which there is a deletion in every segment. The mutant embryos normally fail to hatch but by in vivo culture we were able to show which could produce adult structures. Cultured wg⁻ embryos did not produce any adult structures. Cultured en⁻ embryos produced eye-antennal derivatives and rarely produced partial thoracic structures. nkd⁻ and ptc⁻ embryos produced eye-antennal and thoracic derivatives. The nkd⁻ and ptc⁻ thoracic imaginal discs developed with an abnormal morphology and abnormal pattern of en-expression. Our findings are consistent with the idea that the thoracic imaginal discs derive from two adjacent groups of cells that express wg and en respectively in the embryo.

Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity.

We have examined the developmental consequences for larval and imaginal segmental cuticular structure of a chromosomal translocation involving a breakpoint in the abdominal region of the bithorax complex (BX-C). This complex makes an essential contribution to the development of metameric differences in part of the thorax and in all abdominal segments. The breakpoint is proximal to the most distal (iab-7) homeobox, and results in the translocation to the Y chromosome of the Ultrabithorax (Ubx) and abdominal-A (abd-A) domains. The genotype deficient for the distal part of the complex shows normal function for Ubx and abd-A but has a phenotype typical for severe Abd-B mutations. Conversely, the distal fragment retains a segment identity function which must represent a contribution from Abd-B in parasegments 13 and 14; the latter metamere is wild type, indicating that it does not require the contribution of Ubx or abd-A. We also constructed a genotype comprising the proximal fragment of this translocation together with an overlapping distal fragment of the BX-C derived from Df(3R)Ubx109. It therefore contained all sequences of the BX-C though in the abdominal region the abd-A and Abd-B domains were not adjacent to each other in the chromosome. This genotype was phenotypically normal and demonstrates that DNA sequences in the abd-A and Abd-B regions do not require cis-arrangement for their activity.

The effect of lethal mutations and deletions within the bithorax complex upon the identity of caudal metameres in the Drosophila embryo.

Mutations and deletions of the abdA and AbdB functions in the bithorax complex of Drosophila melanogaster have been examined for their effect upon the hypodermal derivatives of the caudal segments of the embryo, employing light- and scanning electron microscopy. No cuticular structures located posterior to the denticle belt of abdominal segment 8 are affected in abdA- embryos. Embryos of AbdB- genotype no longer have six of the seven pairs of sense organs present in this region, lack posterior spiracles but instead have sclerotized cuticle and sense organs typical of the head region and a rudimentary extra ventral denticle belt. The anal pads, tuft and sense organ 1 do not require BX-C functions for their specification. We discuss the provenance of these cuticular structures and the domain of function of elements within the bithorax complex in terms of parasegmental metameric units.

Recessive lethal mutations within the bithorax-complex in Drosophila.

Genetic deficiencies of the bithorax-complex (BX-C) in Drosophila, have been used to recover recessive lethal mutations in this chromosome region following mutagenesis. Complementation analysis separates these lethal mutations into five groups within a smaller deficiency, thought to remove the entire BX-C, and into 20 to the left and 4 to the right of the region. Homozygotes for each of only three groups of lethals, Ubx, abdA and AbdB, produce homoeotic segmental transformations in embryos. The functional domains of abdA and AbdB have been defined by changes in the appearance of larval hypodermal structures and of clones in imaginal tissue. The function abdA is required in all the compartments caudal to the anteroposterior border of abdominal segment 1 up to and including the anterior region of abdominal segment 8, whilst AbdB is required in abdominal segments 5 to 9. One allele of AbdB produces a ninth abdominal setal band and structures characteristic of head segments posterior to A8. Rare adult survivors hemizygous for an AbdB allele have eight abdominal segments in both sexes, and lack genitalia in females. Our findings are discussed in the context of the organisation of genetic functions within the BX-C.