Kinetics of Plasma Cell-Free DNA and Creatine Kinase in a Canine Model of Tissue Injury.

BACKGROUND: Cell-free DNA (cfDNA) comprises short, double-stranded circulating DNA sequences released from damaged cells. In people, cfDNA concentrations correlate well with disease severity and tissue damage. No reports are available regarding cfDNA kinetics in dogs. OBJECTIVES/HYPOTHESIS: Cell-free DNA will have a short biological half-life and would be able to stratify mild, moderate, and severe tissue injury. Our study aims were to determine the kinetics and biological half-life of cfDNA and to contrast them with those of creatine kinase (CK). ANIMALS: Three groups of 10 dogs undergoing open ovariohysterectomy, surgery for cranial cruciate ligament rupture (CCLR), or hemilaminectomy. METHODS: Plasma for cfDNA and CK analysis was collected at admission, at induction of anesthesia, postsurgery (time 0) and at 6, 12, 24, 36, 48, 60, and 72 hours after surgery. RESULTS: The biological half-life of plasma cfDNA and CK were 5.64 hours (95% confidence interval [CI 95], 4.36-7.98 hours) and 28.7 hours (CI95, 25.3-33.3 hours), respectively. In the hemilaminectomy group, cfDNA concentrations differed significantly from admission at 6-12 hours after surgery. Creatine kinase activity differed among the surgical groups and reached a peak 6 hours after surgery. In the ovariohysterectomy and CCLR groups, plasma CK activity 72 hours after surgery did not differ from admission activity of the ovariohysterectomy group. In contrast, in the hemilaminectomy group, plasma CK activity after 72 hours did not return to the ovariohysterectomy group admission activity. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma CK activity has a longer biological half-life than previously thought. In contrast to plasma CK activity, cfDNA has a short half-life and could be a useful marker for peracute severe tissue injury.

Complement polymorphisms: geographical distribution and relevance to disease.

The evolution of man has been characterised by recurrent episodes of migration and settlement with infectious disease a constant threat. This long history of demographic change, together with the action of evolutionary forces such as natural selection and genetic drift, has shaped human genetic diversity. In particular, the interaction between humans, pathogens and the environment has played a crucial role in generating patterns of human genetic variation. The complement system plays a crucial role in the early protective immune response after exposure to a pathogen. Pathogens, over time, have developed mechanisms to circumvent the effects of complement which in turn has led to development of a more complex complement system. During the evolution of the complement system genes coding complement proteins have evolved polymorphisms, some of which have a functional effect, and this may reflect human-pathogen interaction and geographical origin. An example is the polymorphism Ile62Val (rs800292 (A>G)) in the complement regulator Factor H gene which alters the susceptibility to age-related macular degeneration (AMD), with the Ile62 polymorphism protecting against AMD. When sub-Saharan African and European populations are compared, the frequency of this polymorphism shows a very marked geographical distribution. Polymorphisms in other complement genes such as complement factor B show similar trends. This paper describes the geographical variation present in complement genes and discusses the implications of these observations. The analysis of genetic variation in complement genes is a promising tool to unravel mechanisms of host-pathogen interaction and can provide new insights into the evolution of the human immune system.

Assignment of paternity groups without access to parental genotypes: multiple mating and developmental plasticity in squid.

We present a novel approach to investigating sibling relationships and reconstructing parental genotypes from a progeny array. The Bayesian method we have employed is flexible and may be applicable to a variety of situations in addition to the one presented here. While mutation rates and breeding population allele frequencies can be taken into account, the model requires relatively few loci and makes few assumptions. Paternity of 270 veined squid (Loligo forbesi) hatchlings from three egg strings collected from one location was assigned using five microsatellite loci. Paternal and maternal genotypes reconstructed for each of the three strings were identical, strongly indicating that a single female produced the strings that were fertilized by the same four males. The proportion of eggs fertilized was not equal between males in all three strings, with male 1 siring most offspring (up to 68% in string 1), through to male 4 siring the least (as low as 2.4% in string 1). Although temperature had a profound effect on incubation time, paternity did not affect this trait at 12 degrees C or 8 degrees C.

Assessment of mechanism of acquired skewed X inactivation by analysis of twins.

Skewed X-chromosome inactivation in peripheral blood granulocytes becomes more frequent with increasing age, affecting up to half of those over 75 years old. To investigate the mechanisms underlying this phenomenon, X-inactivation profiles in 33 monozygotic and 22 dizygotic elderly twin pairs were studied. Differential methylation-sensitive restriction enzyme cutting at a hypervariable locus in the human androgen receptor gene (HUMARA) was studied on purified granulocytes using T cells as controls. A large genetic effect on skewed granulocytic X inactivation was shown (P <.05); heritability was estimated to be 0.68. A minor part (SD.0151 relative allele frequency [ie, larger/smaller] units) of the observed variance is due to experimental error. A further contributor to acquired skewing is stochastic asymmetric stem cell division, which was modeled and shown as unlikely to account for a substantial part of variance. Two monozygotic twin pairs had X-inactivation ratios skewed markedly in opposite directions, evidence for a further stochastic mechanism, suggestive of a single overrepresented clone. In conclusion, all 3 suggested mechanisms contribute to acquired X inactivation but the dominant mechanism is genetic selection. The observed proportion of putatively clonal hematopoiesis is similar to the lifetime incidence of hematopoietic stem cell malignancy consistent with the concept that clonal hematopoiesis precedes stem cell malignancy.

Inferring population history from microsatellite and enzyme data in serially introduced cane toads, Bufo marinus.

Much progress has been made on inferring population history from molecular data. However, complex demographic scenarios have been considered rarely or have proved intractable. The serial introduction of the South-Central American cane toad Bufo marinus in various Caribbean and Pacific islands involves four major phases: a possible genetic admixture during the first introduction, a bottleneck associated with founding, a transitory population boom, and finally, a demographic stabilization. A large amount of historical and demographic information is available for those introductions and can be combined profitably with molecular data. We used a Bayesian approach to combine this information with microsatellite (10 loci) and enzyme (22 loci) data and used a rejection algorithm to simultaneously estimate the demographic parameters describing the four major phases of the introduction history. The general historical trends supported by microsatellites and enzymes were similar. However, there was a stronger support for a larger bottleneck at introductions for microsatellites than enzymes and for a more balanced genetic admixture for enzymes than for microsatellites. Very little information was obtained from either marker about the transitory population boom observed after each introduction. Possible explanations for differences in resolution of demographic events and discrepancies between results obtained with microsatellites and enzymes were explored. Limits of our model and method for the analysis of nonequilibrium populations were discussed.

Recent male-mediated gene flow over a linguistic barrier in Iberia, suggested by analysis of a Y-chromosomal DNA polymorphism.

We have examined the worldwide distribution of a Y-chromosomal base-substitution polymorphism, the T/C transition at SRY-2627, where the T allele defines haplogroup 22; sequencing of primate homologues shows that the ancestral state cannot be determined unambiguously but is probably the C allele. Of 1,191 human Y chromosomes analyzed, 33 belong to haplogroup 22. Twenty-nine come from Iberia, and the highest frequencies are in Basques (11%; n=117) and Catalans (22%; n=32). Microsatellite and minisatellite (MSY1) diversity analysis shows that non-Iberian haplogroup-22 chromosomes are not significantly different from Iberian ones. The simplest interpretation of these data is that haplogroup 22 arose in Iberia and that non-Iberian cases reflect Iberian emigrants. Several different methods were used to date the origin of the polymorphism: microsatellite data gave ages of 1,650, 2,700, 3,100, or 3,450 years, and MSY1 gave ages of 1,000, 2,300, or 2,650 years, although 95% confidence intervals on all of these figures are wide. The age of the split between Basque and Catalan haplogroup-22 chromosomes was calculated as only 20% of the age of the lineage as a whole. This study thus provides evidence for direct or indirect gene flow over the substantial linguistic barrier between the Indo-European and non-Indo-European-speaking populations of the Catalans and the Basques, during the past few thousand years.

Inbreeding of bottlenecked butterfly populations. Estimation using the likelihood of changes in marker allele frequencies.

Polymorphic enzyme and minisatellite loci were used to estimate the degree of inbreeding in experimentally bottlenecked populations of the butterfly, Bicyclus anynana (Satyridae), three generations after founding events of 2, 6, 20, or 300 individuals, each bottleneck size being replicated at least four times. Heterozygosity fell more than expected, though not significantly so, but this traditional measure of the degree of inbreeding did not make full use of the information from genetic markers. It proved more informative to estimate directly the probability distribution of a measure of inbreeding, sigma2, the variance in the number of descendants left per gene. In all bottlenecked lines, sigma2 was significantly larger than in control lines (300 founders). We demonstrate that this excess inbreeding was brought about both by an increase in the variance of reproductive success of individuals, but also by another process. We argue that in bottlenecked lines linkage disequilibrium generated by the small number of haplotypes passing through the bottleneck resulted in hitchhiking of particular marker alleles with those haplotypes favored by selection. In control lines, linkage disequilibrium was minimal. Our result, indicating more inbreeding than expected from demographic parameters, contrasts with the findings of previous (Drosophila) experiments in which the decline in observed heterozygosity was slower than expected and attributed to associative overdominance. The different outcomes may both be explained as a consequence of linkage disequilibrium under different regimes of inbreeding. The likelihood-based method to estimate inbreeding should be of wide applicability. It was, for example, able to resolve small differences in sigma2 among replicate lines within bottleneck-size treatments, which could be related to the observed variation in reproductive viability.

Genealogical inference from microsatellite data.

Ease and accuracy of typing, together with high levels of polymorphism and widespread distribution in the genome, make microsatellite (or short tandem repeat) loci an attractive potential source of information about both population histories and evolutionary processes. However, microsatellite data are difficult to interpret, in particular because of the frequency of back-mutations. Stochastic models for the underlying genetic processes can be specified, but in the past they have been too complicated for direct analysis. Recent developments in stochastic simulation methodology now allow direct inference about both historical events, such as genealogical coalescence times, and evolutionary parameters, such as mutation rates. A feature of the Markov chain Monte Carlo (MCMC) algorithm that we propose here is that the likelihood computations are simplified by treating the (unknown) ancestral allelic states as auxiliary parameters. We illustrate the algorithm by analyzing microsatellite samples simulated under the model. Our results suggest that a single microsatellite usually does not provide enough information for useful inferences, but that several completely linked microsatellites can be informative about some aspects of genealogical history and evolutionary processes. We also reanalyze data from a previously published human Y chromosome microsatellite study, finding evidence for an effective population size for human Y chromosomes in the low thousands and a recent time since their most recent common ancestor: the 95% interval runs from approximately 15, 000 to 130,000 years, with most likely values around 30,000 years.

Sex differences in coagulation and fibrinolysis in subjects with coronary artery disease.

Women with coronary artery disease (CAD) have a prognosis at least as bad and possibly worse than men. Differences in classical risk factors do not fully account for these findings and there is evidence that circulating levels of haemostatic factors may predict CAD risk. In this study sex differences in haemostatic risk factors were examined in relation to coronary stenosis. 609 (420 men, 69%) subjects admitted for coronary angiography for suspected CAD were recruited. Levels of Factor VII:C (FVII:C), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) were estimated in 296 subjects from one centre. Of these, women (n = 107) had higher levels of FVII:C (134% vs 117%, p < 0.0005), and fibrinogen (3.4 g/l vs 3.2 g/l p = 0.01) than men (n = 189) and these differences remained after adjusting for other covariates. In subjects with angiographically significant atheroma these differences in haemostatic factors (n = 50 for women vs n = 147 for men) were exaggerated, (FVII:C 139% vs 117, p < 0.0001, fibrinogen 3.7 g/l vs 3.3 g/l p = 0.003), PAI-1 (26.2 ng/ml vs 19.7 ng/ml, p = 0.02) with a trend towards higher levels of vWF in the women. Women with significant atheroma at angiography (n = 50) had higher levels of PAI-1 (25.0 ng/ml vs 13.4 ng/ml p < 0.0001) and vWF (1.25 IU/ml vs 1.06 IU/ml, p = 0.02) and a trend towards higher levels of both fibrinogen and FVII:C than women with normal or in significant coronary vessel disease (n = 57). Elevated circulating levels of PAI-1, vWF, fibrinogen and FVII:C in women with angiographically proven CAD may contribute to an adverse cardiovascular risk factor profile and the poorer prognosis in females than male patients with proven coronary artery disease.

Association of the platelet Pl(A) polymorphism of glycoprotein IIb/IIIa and the fibrinogen Bbeta 448 polymorphism with myocardial infarction and extent of coronary artery disease.

BACKGROUND: Platelets and fibrinogen play an integral role in the development of thrombosis and are implicated in the process of atherosclerosis. The fibrinogen Bbeta 448 polymorphism and the Pl(A) polymorphism of platelet glycoprotein IIIa are reported to be independently associated with coronary artery disease. The aim of this study was to determine the association of the fibrinogen Bbeta 448 and the platelet glycoprotein IIIa Pl(A) polymorphisms in relation to extent of coronary atheroma as characterized by angiography and a past history of myocardial infarction (MI) as assessed by World Health Organization criteria. METHODS AND RESULTS: Caucasian patients (n=405) admitted for routine angiography for investigation of chest pain or suspected coronary artery disease were recruited. Caucasian control subjects (n=216) were recruited from local Family Health Services Authority general practice registers. Fibrinogen levels were higher (P=.04) in male patients (3.24 g/L; CI, 3.14 to 3.35) than male control subjects (3.06 g/L; CI, 2.91 to 3.21). There was a trend toward a difference (P=.06) in fibrinogen genotype distributions between female patients (1/1=93, 1/2=31, and 2/2=1) and female control subjects (1/1=67, 1/2=34, and 2/2=5). In logistic regression models the Pl(A2) genotype was associated with MI (odds ratio, 1.66; CI, 1.15 to 2.39; P=.007) and stenosis of more than one vessel (odds ratio, 1.5; CI, 1.01 to 2.26; P=.04). In men suffering an MI before the age of 47 years there was a 50% incidence of the Pl(A2) allele (P=.05), and in these subjects there was evidence of an interaction with cholesterol (P=.04). CONCLUSIONS: We found evidence of an association of the Pl(A2) polymorphism in MI and multiple-vessel stenosis. The association with MI was strongest in young men, in whom there was also evidence of an interaction with cholesterol.

Plasminogen activator inhibitor-1 promoter 4G/5G genotype and plasma levels in relation to a history of myocardial infarction in patients characterized by coronary angiography.

To investigate the relationship between an insertion/deletion (4G/5G) polymorphism in the promoter region of the plasminogen activator inhibitor-1 (PAI-1) gene and the phenotypes of PAI-1 levels, coronary atheroma, and a past history of coronary thrombosis, we studied 453 patients (320 men and 133 women) characterized by coronary angiography. Patients were classified as having normal vessels (n = 125) or single-vessel (n = 92) or multivessel (n = 232) coronary disease on the basis of > or = 50% stenosis. PAI-1 antigen levels were highest in patients with the 4G/4G genotype (22.5 ng/mL), with a stepwise decrease in levels as the number of 4G alleles decreased (21.5 ng/mL for 4G/5G and 15.8 ng/mL for 5G/5G, P = .02) after adjusting for age, sex, triglyceride levels, and body mass index (BMI). The association between triglyceride level and PAI-1 was genotype specific, with a steeper slope in subjects with the 4G/4G genotype (P = .004). A gene-environment interaction between BMI, PAI-1, and genotype was observed, with a steeper association in patients with the 5G/5G genotype (P = .02). The 4G/4G genotype was significantly associated with a history of myocardial infarction (P < .03; odds ratio, 2.0; 95% CI, 1.1 to 3.7). This relationship was stronger in subjects with diseased vessels (P = .006). There was no relationship between either PAI-1 genotype or levels and the presence of atheroma. Our data suggest that PAI-1 promoter polymorphism influences the development of myocardial infarction through its effect on thrombus formation in patients with preexisting coronary atheroma.

Double-pass oblique-incidence interferometer for the inspection of nonoptical surfaces.

Interferometric testing of the flatness or straightness of ground surfaces of metals, granite, ceramics, and glasses has always proved difficult because of the extremely low specular reflection from these optically rough surfaces. At oblique angles of incidence the specular reflection coefficient of nonoptical surfaces increases rapidly, typically from near 0.01 at 75 degrees up to, say, 0.50 at 85 degrees and near 1.00 at 90 degrees angle of incidence. By taking advantage of this enhanced reflection at oblique incidence and the brightness and spatial coherence of visible gas lasers, it becomes practical to use interferometric inspection of nonoptical surfaces. A novel double-pass oblique-incidence interferometer is described which enables testing of large pieces having a specular reflection as low as 1%.

Anomalous right coronary artery arising from left mainstem.

Various coronary artery anomalies occur in both symptomatic and asymptomatic individuals. We have described a unique case of an aberrant right coronary artery arising from the left mainstem, resulting in clinical myocardial infarction in the absence of coronary atherosclerosis. Though different anomalies of the right coronary artery have been described, we feel this case is unique in that the right coronary artery arises from the left mainstem, truly forming a single coronary artery.

Groove depth determination using a laser for sinusoidal groove gratings.

We describe a technique for determining the groove depth of gratings having sinusoidal grooves. Absolute efficiencies measured using a laser are compared with numerical results to give an indication of groove depth.