Early or late pregnancy loss and development of clinical cardiovascular disease risk factors: a prospective cohort study.

OBJECTIVE: To assess the association between the outcome of a woman's first pregnancy and risk of clinical cardiovascular disease risk factors. DESIGN: Prospective cohort study. SETTING AND POPULATION: Nurses' Health Study II. METHODS: Multivariable-adjusted Cox proportional hazards models were used to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations between first pregnancy outcome and hypertension, type 2 diabetes, and hypercholesterolemia. MAIN OUTCOME MEASURES: Hypertension, type 2 diabetes, and hypercholesterolemia. RESULTS: Compared to women who reported a singleton live first birth, women with early spontaneous abortion (<12 weeks) had a greater rate of type 2 diabetes (HR: 1.20; 95% CI: 1.07-1.34) and hypercholesterolemia (HR: 1.06; 95% CI: 1.02-1.10), and a marginally increased rate of hypertension (HR: 1.05, 95% CI: 1.00-1.11). Late spontaneous abortion (12-19 weeks) was associated with an increased rate of type 2 diabetes (HR: 1.38; 95% CI: 1.14-1.65), hypercholesterolemia (HR: 1.11; 95% CI: 1.03-1.19), and hypertension (HR: 1.15; 95% CI: 1.05-1.25). The rates of type 2 diabetes (HR: 1.45; 95% CI: 1.13-1.87) and hypertension (HR: 1.15; 95% CI: 1.01-1.30) were higher in women who delivered stillbirth. In contrast, women whose first pregnancy ended in an induced abortion had lower rates of hypertension (HR: 0.87; 95% CI: 0.84-0.91) and type 2 diabetes (HR: 0.89; 95% CI: 0.79-0.99) than women with a singleton live birth. CONCLUSIONS: Several types of pregnancy loss were associated with an increased rate of hypertension, type 2 diabetes, and hypercholesterolemia, which may provide novel insight into the pathways through which pregnancy outcomes and CVD are linked. TWEETABLE ABSTRACT: Pregnancy loss is associated with later maternal risk of hypertension, type 2 diabetes, and hypercholesterolemia.

Pivoting from Arabidopsis to wheat to understand how agricultural plants integrate responses to biotic stress.

In this review, we argue for a research initiative on wheat's responses to biotic stress. One goal is to begin a conversation between the disparate communities of plant pathology and entomology. Another is to understand how responses to a variety of agents of biotic stress are integrated in an important crop. We propose gene-for-gene interactions as the focus of the research initiative. On the parasite's side is an Avirulence (Avr) gene that encodes one of the many effector proteins the parasite applies to the plant to assist with colonization. On the plant's side is a Resistance (R) gene that mediates a surveillance system that detects the Avr protein directly or indirectly and triggers effector-triggered plant immunity. Even though arthropods are responsible for a significant proportion of plant biotic stress, they have not been integrated into important models of plant immunity that come from plant pathology. A roadblock has been the absence of molecular evidence for arthropod Avr effectors. Thirty years after this evidence was discovered in a plant pathogen, there is now evidence for arthropods with the cloning of the Hessian fly's vH13 Avr gene. After reviewing the two models of plant immunity, we discuss how arthropods could be incorporated. We end by showing features that make wheat an interesting system for plant immunity, including 479 resistance genes known from agriculture that target viruses, bacteria, fungi, nematodes, insects, and mites. It is not likely that humans will be subsisting on Arabidopsis in the year 2050. It is time to start understanding how agricultural plants integrate responses to biotic stress.

A reproductive fitness cost associated with Hessian fly (Diptera: Cecidomyiidae) virulence to wheat's H gene-mediated resistance.

We studied whether adaptation of the Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), to plant resistance incurs fitness costs. In this gene-for-gene interaction, adaptation to a single H resistance gene occurs via loss of a single effector encoded by an Avirulence gene. By losing the effector, the adapted larva now survives on the H gene plant, presumably because it evades the plant's H gene-mediated surveillance system. The problem is the Hessian fly larva needs its effectors for colonization. Thus, for adapted individuals, there may be a cost for losing the effector, with this then creating a trade-off between surviving on H-resistant plants and growing on plants that lack H genes. In two different tests, we used wheat lacking H genes to compare the survival and growth of a nonadapted strain to two H-adapted strains. The two adapted strains differed in that one had been selected for adaptation to H9, whereas the other strain had been selected for adaptation to H13. Tests showed that two H-adapted strains were similar to the nonadapted strain in egg-to-adult survival but that they differed in producing adults with smaller wings. By using known relationships between wing length and reproductive potential, we found that losses in wing length underestimate losses in reproductive potential. For example, H9- and H13-adapted females had 9 and 3% wing losses, respectively, but they were estimated to have 32 and 12% losses in egg production. Fitness costs of adaptation will be investigated further via selection experiments comparing Avirulence allele frequencies for Hessian fly populations exposed or not exposed to H genes.

Assessment of structural variation and molecular mapping of insertion sites of Desmar-like elements in the Hessian fly genome.

The Hessian fly (Mayetiola destructor) is an agriculturally important pest of wheat. A mariner element (Desmar1) has been previously identified in the Hessian fly genome. Using Desmar1 as a probe, we isolated individual copies of Desmar-like elements from the Hessian fly genome cloned in bacterial artificial chromosomes (BACs) and studied their structural variability and flanking DNA sequences. The partial Desmar-like copies are relatively more abundant (∼64%) than full length copies (∼36%) in the Hessian fly genome. Most of the full length copies are consistently flanked by an EcoRI restriction site that occurs 32 bp from one end and 66 bp from the other end of the mariner. Using an amplified fragment length polymorphism-PCR (AFLP-PCR) based method, we identified segregating polymorphisms associated with Desmar elements in a F2 mapping population. We were able to use the segregation data to localize the chromosomal position of three Desmar elements by linkage analysis. As paternal chromosomes are eliminated in the Hessian fly during early embryogenesis, two-thirds of the AFLPs were expected to be polymorphic in the mapping population and this was observed for AFLPs anchored to full length Desmar copies but not to the partial copies. Thus, our data indicate that dead and partial Desmar-like copies are probably associated with less polymorphic regions and may represent mariner graveyards in the Hessian fly genome.

Tick genomics: the Ixodes genome project and beyond.

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.

Molecular cloning and characterization of two digestive serine proteases from the Hessian fly, Mayetiola destructor.

Full-length cDNA and genomic sequences for two genes (designated mdesprot-I and mdesprot-II) encoding digestive serine proteases in Hessian fly, Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Both mdesprot-I and mdesprot-II encoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescent in situ hybridization revealed the cytological localization of mdesprot-I and mdesprot-II on the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches.

Expression of Cyp6g1 and Cyp12d1 in DDT resistant and susceptible strains of Drosophila melanogaster.

The Rst(2)DDT locus (loci) in Drosophila is associated with the over-expression of two cytochrome P450 genes, Cyp6g1 and Cyp12d1. Using northern and western blot analysis we observed the expression pattern of these two genes in two DDT susceptible (Canton-S and 91-C) and three DDT resistant strains (Wisconsin, 91-R and Hikone-R). In Canton-S and 91-R, the CYP6G1 protein was constitutively expressed throughout development. In the Wisconsin strain, CYP6G1 was not expressed in third instar larvae unless the larvae are exposed to DDT. CYP12D1 protein was only expressed in adults. Cyp12d1 mRNA is induced in DDT resistant strains post-exposure to DDT and the expression patterns of Cyp12d1 mRNA varied across DDT resistant strains. Our data support the hypothesis that there is evolutionary plasticity in the expression patterns of P450s associated with metabolic pesticide resistance.

A group of related cDNAs encoding secreted proteins from Hessian fly [Mayetiola destructor (Say)] salivary glands.

A group of cDNAs has been isolated and characterized from Hessian fly [Mayetiola destructor (Say)] salivary glands. Members in this group appear to encode proteins with secretion signal peptides at the N-terminals. The mature putative proteins are small, basic proteins with calculated molecular weights that ranged from 8.5 to 10 kDa, and isoelectric points from 9.92 to 10.90. Sequence analysis indicated a strong selection for mutations that generate amino acid changes within the coding region. Northern blot analysis revealed that these genes are expressed only in the first instar larvae, a critical stage that determines if the interaction between a specific Hessian fly biotype and a specific wheat cultivar is compatible. Genomic analysis demonstrated that multiple copies of similar genes are clustered within a short region on chromosome 2A. This is the same arm in which two avirulence genes have been mapped.

Grasses and gall midges: plant defense and insect adaptation.

The interactions of two economically important gall midge species, the rice gall midge and the Hessian fly, with their host plants, rice and wheat, respectively, are characterized by plant defense via R genes and insect adaptation via avr genes. The interaction of a third gall midge species, the orange wheat blossom midge, with wheat defense R genes has not yet exhibited insect adaptation. Because of the simple genetics underlying important aspects of these gall midge-grass interactions, a unique opportunity exists for integrating plant and insect molecular genetics with coevolutionary ecology. We present an overview of some genetic, physiological, behavioral, and ecological studies that will contribute to this integration and point to areas in need of study.

The 3' UTR of human MnSOD mRNA hybridizes to a small cytoplasmic RNA and inhibits gene expression.

Human MnSOD localizes to the mitochondria and plays a key protective role by detoxifying oxygen free radicals. The MnSOD mRNA 3' UTR contains a 280-bp region (Alu-like element or Alu-E) that shows high homology to human Alu and 7SL sequences. MnSOD 3' UTR probes hybridize to a specific cytoplasmic RNA species of approximately 300 nucleotides. This antisense RNA is most likely 7SL RNA based on its size, ubiquitousness, high levels, and lack of inducibility. Hybridization of this small RNA to the MnSOD 3' UTR may modulate posttranscriptional MnSOD gene expression. This regulation could occur by several means including inhibition of translation and mRNA destabilization. Regulation at the level of translational initiation does not seem to occur as MnSOD mRNA containing the Alu-E is efficiently bound by ribosomes. To test the role of the MnSOD 3' UTR, and in particular the Alu-E in gene expression, luciferase reporter gene constructs were made containing various regions of the MnSOD 3' UTR including the Alu-E. These constructs were transfected into human A549 lung carcinoma cells and luciferase activity was measured. Reporter constructs containing the MnSOD 3' UTR and the Alu-E repress luciferase activity. Taken together, these results suggest that naturally occurring antisense RNA may bind MnSOD mRNA and repress its expression. These results also suggest that other mRNAs containing Alu elements may be similarly repressed.

Molecular genetic mapping of three X-linked avirulence genes, vH6, vH9 and vH13, in the Hessian fly.

Three X-linked avirulence genes, vH6, vH9, and vH13 in the Hessian fly, Mayetiola destructor, confer avirulence to Hessian fly resistance genes H6, H9, and H13 in wheat. We used a combination of two- and three-point crosses to determine the order of these genes with respect to each other, the white eye mutation and three X-linked molecular markers, G15-1, 020, and 021, developed from genomic lambda clones, lambda G15-1, lambda 020, and lambda 021. The gene order was determined to be vH9-vH6-G15-1-w-vH13-020-021. In situ hybridization of lambda G15-1, lambda 020, and lambda 021, on the polytene chromosomes of the Hessian fly salivary gland established their orientation on Hessian fly chromosome X1. Based on the size of the Hessian fly genome, and the genetic distances between markers, the relationship of physical to genetic distance was estimated at no more than 300 kb/cM along Hessian fly chromosome X1, suggesting that map-based cloning of these avirulence genes will be feasible.

Genetic mapping of Hessian fly avirulence gene vH6 using bulked segregant analysis.

The Hessian fly, Mayetiola destructor (Say), an important insect pest of wheat, Triticum aestivum L., has a gene-for-gene relationship with wheat: single genes in the insect condition avirulence to specific resistance genes in wheat. We report the discovery of the first molecular genetic marker that is tightly linked to a Hessian fly avirulence gene. This dominant DNA polymorphism (OPG15-1) was identified using bulked segregant analysis and arbitrary primers in polymerase chain reactions. Bulked segregant analysis was modified to accommodate the anomalous chromosome cycle of the Hessian fly. It was used to identify DNA polymorphisms linked to the gene (vH6) that confers avirulence to the resistance gene H6 in wheat. OPG15-1 was cloned and sequenced, and a pair of site-specific primers were designed that converted it into a codominant single-stranded conformational polymorphism. Both OPG15-1 and vH6 were shown to be X-linked, and the genetic distance between the two loci was 2.5 +/- 2.5 cM. In situ hybridization to polytene chromosomes of larval salivary glands indicated that OPG15-1 resides near the centromere of Hessian fly chromosome X1.

Genetic mapping of a major locus controlling pyrethroid resistance in Tribolium castaneum (Coleoptera: Tenebrionidae).

Tribolium castaneum (Herbst) strain QTC279 is highly resistant to deltamethrin and other synthetic pyrethroids. This strain was shown to carry at least 1 resistance gene, PyR-1, on linkage group 9, approximately 20 map units from the visible mutant marker, pearl. Three-point mapping involving pearl and another visible mutant marker, cola, indicated a gene order of pearl-cola-PyR-1. Evidence of a 2nd LG9-linked resistance factor (R) mapping in the gene order R-p-co was also observed. Other resistance factors were clearly present in QTC279, but were not genetically mapped. Piperonyl butoxide, an inhibitor of cytochrome P450-mediated oxidative metabolism, significantly increased the toxicity of deltamethrin to a strain derived from QTC279 that carries PyR-1, strain pR. Compared to susceptible beetles, QTC279 and pR had elevated and comparable levels of cytochrome P450 protein. The significance of pyrethroid resistance in T. castaneum is discussed.

Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum.

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.

Chromosome extraction and revision of linkage group 2 in Tribolium castaneum.

We used a balancer chromosome to recover ethylmethanesulfonate-induced recessive mutations in a targeted region of the genome of the red flour beetle (Tribolium castaneum) by the technique of chromosome extraction. The experiments reported herein constitute the first successful application of this powerful technique in the order Coleoptera. Using the balancer chromosome maxillopedia-Dachs3 (mxpDch-3), we recovered seven recessive visible variants representing seven distinct loci and several dozen recessive lethal variants representing at least five distinct loci after screening 1,607 EMS-mutagenized chromosomes. A subset of the mxpDch-3-extracted mutations were positioned on the map of the second linkage group by a series of two-, three-, and four-point crosses. The orientation of the homeotic gene complex (HOM-C) on this linkage group was also determined. With the advent of better and more varied balancer chromosomes and the concomitant improvement of chromosome extraction procedures for genetic analysis of T. castaneum, saturation mutagenesis of targeted regions of the genome is now feasible in this species.

Cytogenetics of chromosome rearrangements in Tribolium castaneum.

The karyotype of the red flour beetle, Tribolium castaneum, was reexamined and improved by restriction enzyme banding with HpaII. After this treatment, each of the 10 chromosomes were identified in spermatogonial metaphase cells and 3 of the 8 autosomal bivalents and the XY pair were identified in spermatocyte metaphase I nuclei. Based on centromere position, relative length, and banding pattern, probable correlations between some of the mitotic chromosomes and some of the metaphase I bivalents were ascertained. Thus improved, the karyotypes of beetles harboring genetically defined translocations were investigated. Spermatocyte metaphase I nuclei were most informative, as normal chromosome pairing was visibly disrupted by rearrangements. Bivalents associated with each rearrangement were identified. Results demonstrated that each of the five best defined T. castaneum linkage groups corresponds to a different chromosome and established correspondence between bivalents and linkage groups 1-4. The relevance of these findings is discussed with regard to Tribolium genetics and evolution.

Predictive value of blood clotting tests in cardiac surgical patients.

This study prospectively evaluated numerous tests of clotting function in 897 consecutive adult cardiac surgical patients over 18 months. This included coronary operation, valve replacement, and reoperative patients. The tests included activated clotting time, activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen, fibrin/fibrinogen degradation products, platelet count, and Duke's earlobe bleeding time. Other variables such as age, sex, and cardiopulmonary bypass duration were included in the multivariate analysis. Statistically significant correlations were found between 16-hour mediastinal drainage and activated partial thromboplastin time, fibrinogen, activated clotting time, fibrin/fibrinogen degradation products, platelet count, and prothrombin time. Scatter plots indicate that these relationships, although statistically significant, had little predictive value and were largely significant as a result of the large number of patients in each group, which permitted weak correlations to reach statistical significance. The best multivariate model constructed could explain only 12% of the observed variation in postoperative blood loss. Because the predictive values of the tests are so low, it does not appear sensible to screen patients routinely using these clotting tests shortly after cardiopulmonary bypass.

Structure and function of the homeotic gene complex (HOM-C) in the beetle, Tribolium castaneum.

The powerful combination of genetic, developmental and molecular approaches possible with the fruit fly, Drosophila melanogaster, has led to a profound understanding of the genetic control of early developmental events. However, Drosophila is a highly specialized long germ insect, and the mechanisms controlling its early development may not be typical of insects or Arthropods in general. The beetle, Tribolium castaneum, offers a similar opportunity to integrate high resolution genetic analysis with the developmental/molecular approaches currently used in other organisms. Early results document significant differences between insect orders in the functions of genes responsible for establishing developmental commitments.

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex.

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.