Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.     

Evolution of the gall wasp-host plant association

Gall wasps, or cynipids, form the second largest radiation of galling insects with more than 1300 described species. According to current views, the first cynipids were phytophagous and developed in herb stems of the Asteraceae without modifying plant growth or development. The first galls were supposedly multichambered stem swellings, and subsequent trends involved increase in gall complexity and reduction in the number of larval chambers. Gall wasps also have many of the features believed to be characteristic for phytophagous insects radiating in parallel with their host plants. We tested these hypotheses by mapping characters onto a recent estimate of higher cynipid relationships from a morphology-based analysis of exemplar taxa, controlling for phylogenetic uncertainty using bootstrapping. Characters were also mapped onto a metatree including all gall wasps, assembled from phylogenetic analyses as well as recent classifications. The results contradict many of the current hypotheses. The first cynipids with extant descendants were not Asteraceae stem feeders but induced distinct single-chambered galls in reproductive organs of herbaceous Papaveraceae, or possibly Lamiaceae. There has been a general trend toward more complex galls but the herb-stem feeders evolved from ancestors inducing distinct galls and their larval chambers are best understood as cryptic galls. Woody hosts have been colonized only three times, making the apparently irreversible transition from herbs to woody hosts one of the most conservative features of the gall wasp-host plant association. The evolution of host plant preferences is characterized by colonization of preexisting host-plant lineages rather than by parallel cladogenesis. Cynipids are mono- or oligophagous and host-plant choice is strongly phylogenetically conserved. Yet, the few major host shifts have involved remarkably distantly related plants. Many shifts have been onto plant species already exploited by other gall wasps, suggesting that interspecific parasitism among cynipids facilitates colonization of novel host plants.     

Taxonomic distinctness and species richness as measures of functional structure in bird assemblages

Most traditional "biodiversity" indices have an uncertain ecological interpretation, unfavourable sampling properties, and excessive data requirements. A new index of taxonomic distinctness (the average evolutionary distance between species in an assemblage) has many advantages over traditional measures, but its ecological interpretation remains unclear. We used published behavioural species data in conjunction with bird atlas data to quantify simple functional metrics (the fraction of species engaged in non-competitive interactions, and the average between-species disparity in habitat preferences) for breeding-bird assemblages in Europe and North America. We then analysed correlations of functional metrics with taxonomic distinctness and species richness, respectively. All functional metrics had weak, positive correlations with species richness. In contrast, correlations between functional metrics and taxonomic distinctness ranged from slightly negative to strongly positive, depending on the relative habitat heterogeneity, and on the resource involved in the between-species interaction. Strong positive correlations between taxonomic distinctness and the fraction of interactive species occurred for resources with few producer species per consumer species, and we suggest that taxonomic distinctness is consistently correlated with conservation worth. With its favourable sampling properties and data requirements, this taxonomic distinctness measure is a promising tool for biodiversity research and for environmental monitoring and management.     

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.     

Inhibition of the L-dopa transport system in human epidermal Langerhans cells by omeprazole and its analogues

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) transport into human Langerhans cells (LC) occurs by a saturable mediation. This plasma membrane agency is, due to its characteristics, distinguishable from systems transporting other neutral, cationic and anionic amino acids into other cells and serves to catalyze the flow of L-dopa, only, into LC. The uphill operation of this L-dopa transport system is believed to occur by down-gradient countermigration of H⁺. Due to the uniqueness of the L-dopa transport system, the widely used analogue inhibition approach was not applicable. Instead we studied omeprazole and its analogues in our search for suitable inhibitory candidates. Omeprazole and most of its analogues were indeed inhibitory in the concentration range 1–100mol/L. Conspicuously, the compounds with strongest polarity were least inhibitory. The inhibitory pattern displayed by omeprazole and the other analogues on L-dopa uptake in LC corresponded to some extent to what has been observed previously for purified H⁺,K⁺-ATPase from tubulovesicles of the stomach. No effects of the inhibitors were registered on energy charge and lactate production of epidermal biopsies, nor were any gross alterations of ultrastructure of LC noticed.     

Mediated exodus of L-dopa from human epidermal Langerhans cells

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, -methyl-dopa (-m-dopa), D-dopa and the dipeptide, met–ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5–7min and a complete exodus of L-dopa was attained at about 20min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of -m-dopa, D-dopa and met–ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons

BACKGROUND AND PURPOSE: Ghrelin is a peptide discovered in endocrine cells of the stomach. Since ghrelin mRNA expression and plasma levels are elevated in the fasting state, we investigated the effects of ghrelin on the interdigestive migrating myoelectric complex (MMC) in the small intestine in vivo and compared with motor effects of ghrelin in vitro. Methods: Sprague-Dawley rats were supplied with a venous catheter and bipolar electrodes in the duodenum and jejunum for electromyography of small intestine in awake rats. In organ baths, isometric contractions of segments of rat jejunum were studied. RESULTS: Ghrelin dose-dependently shortened the MMC cycle length at all three recording points. At the duodenal site, the interval shortened from 17.2+/-2.0 to 9.9+/-0.8 min during infusion of ghrelin (1000 pmol kg(-1) min(-1)) and at the jejunal site from 17.5+/-2.2 to 10.5+/-0.8 min. Ghrelin contracted the muscle strips with a pD2 of 7.97+/-0.47. Atropine (10(-6) M) in vitro and (1 mg kg(-1)) in vivo blocked the effect of ghrelin. CONCLUSION: Ghrelin stimulates interdigestive motility through cholinergic neurons. Ghrelin also stimulates motility, in vitro, suggesting that ghrelin receptors are present in the intestinal neuromuscular tissue and mediate its effects via cholinergic mechanisms.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.