Genotyping by apyrase-mediated allele-specific extension

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3′-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3′-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3′-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.     

Distribution and oviposition preference of galling sawflies in arctic Canada

The distribution and ecology of insects in arctic regions are poorly known. The aim of this study was to determine the distribution of galling sawflies in the Canadian arctic and their oviposition preference. The Swedish Tundra Northwest 1999 expedition visited 17 sites in the Canadian arctic. We determined the occurrence of galling sawflies at all the sites and studied the oviposition preference of two leaf-galling sawflies, Eupontania arctica and Pontania nivalis, on Salix reticulata and S. glauca, respectively. Galling sawflies were abundant at only one site, the mainland site at Ivvavik National Park. Only a few galls in total were found at the remaining sites, suggesting that galling sawflies are rare in the higher arctic, and potential explanations for this pattern are discussed. Shoots with leaf galls were longer than shoots without galls on both S. reticulata and S. glauca. These differences could not be explained by a higher number of leaves on longer shoots. This suggests that long shoots are preferred by sawflies because of faster development and better survival of larvae on long shoots.     

Nuclease-Assisted Suppression of Human DNA Background in Sepsis

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C₀t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C₀t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.     

IgG Suppresses Antibody Responses in Mice Lacking C1q,C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.     

Assessing acid stress in Swedish boreal and alpine streams using benthic macroinvertebrates

Sixty streams in northern Sweden were sampled for benthic macroinvertebrates in spring and autumn of 2000 as part of the European Union project AQEM (the Development and Testing of an Integrated Assessment System for the Ecological Quality of Streams and Rivers throughout Europe using Benthic Macroinvertebrates). Samples were taken using a harmonised multi-habitat sampling procedure and a large number of parameters describing the streams and their catchments were recorded for all sampling sites. From the stream water chemistry characteristics unbiased measures of acid stress were derived, based on the Swedish Ecological Quality Criteria (EQC) for acidity status. These criteria do not, however, assess the degree of human influence to a stream. Therefore, in the following analysis of classification, the Swedish EQC status criteria are applied as if they were a measure of human influence. Three new multimetric acid indices using benthic macroinvertebrates were developed for the AQEM project. These, together with a large number of other benthic macroinvertebrate indices including a number of indices aimed at detecting acid stress, were evaluated for their ability to detect acid stress, using both spring/early summer and autumn sampling. Surprisingly, the acid index that is in general use in northern Sweden worked well in spring/early summer, but because of a (probable) re-colonisation of more acid sensitive taxa, the index values changed from classifying streams as affected in spring/early summer to classify streams as non-affected by acid stress in autumn. Another Swedish acid index (included in the Swedish Environmental Quality Criteria developed by the Swedish EPA) and developed for southern Sweden was strongly correlated between spring/early summer and autumn and could thus be sampled in the autumn and indicate acid stress from spring flood declines in pH. This is of great importance since the local County boards of northern Sweden generally argue that sampling has to be done in the spring to see the effects of acid stress, when sampling is difficult for logistic reasons. Whether or not the three indices developed for the AQEM project (and extensively based on the south Swedish acid index) were truly better than the south-Swedish acid index to assess acid stress in northern Sweden could not be clearly determined in the present study.     

Extensive linkage disequilibrium in small human populations in Eurasia

The extent of linkage disequilibrium (LD) was studied in two small food-gathering populations—Evenki and Saami—and two larger food-producing populations—Finns and Swedes—in northern Eurasia. In total, 50 single-nucleotide polymorphisms (SNPs) from five genes were genotyped using real-time pyrophosphate DNA sequencing, whereas 14 microsatellites were genotyped in two X-chromosomal regions. In addition, hypervariable region I of the mtDNA was sequenced to shed light on the demographic history of the populations. The SNP data, as well as the microsatellite data, reveal extensive levels of LD in Evenki and Saami when compared to Finns and Swedes. mtDNA-sequence variation is compatible with constant population size over time in Evenki and Saami but indicates population expansion in Finns and Swedes. Furthermore, the similarity between Finns and Swedes in SNP allele- and haplotype-frequency distributions indicate that these two populations may share a recent common origin. These findings suggest that populations such as the Evenki and the Saami, rather than the Finns, may be particularly suited for the initial coarse mapping of common complex diseases.     

Triage of women with equivocal or low-grade cervical cytology results: a meta-analysis of the HPV test positivity rate

Consistent evidence underlines the utility of human papillomavirus (HPV) DNA testing in the management of women with equivocal cervical cytological abnormalities, but not in case of low-grade lesions. We performed a meta-analysis including studies where the high-risk probe of the Hybrid Capture-II is used to triage these two cytological categories. The triage test-positivity rate reflects the colposcopy referral workload.Data were pooled on the HPV test positivity rate in women with atypical squamous cells of undetermined significance (ASCUS/ASC-US) or low-grade squamous intraepithelial lesions (LSIL), derived from different cytological classification systems. The meta-analysis was restricted to studies, published between 1991 and 2007. A random-effect model was applied for meta-analytical pooling and the influence of covariates on the HPV positivity rate was analyzed by meta-regression. The variation by age was assessed within individual studies since age strata were not defined uniformly. On an average, 43% (95% CI: 40–46%) of women with ASCUS/ASC-US were high-risk HPV positive (range 23–74%). In women with LSIL, the pooled positivity rate was 76% (95% CI: 71–81%; range 55–89%). In spite of considerable inter-study heterogeneity, the difference in HPV positivity between the two triage groups was large and highly significant: 32% (95% CI: 27–38%). HPV rates dropped tremendously as age and cutoffs of test positivity increased. Other factors (cytological classification system, country, continent, collection method and year of publication) had no statistically significant impact, except in LSIL triage where HPV positivity was significantly lower in European compared to American studies. Women with LSIL, especially younger women, have high HPV positivity rates suggesting limited utility of reflex HPV triaging these cases. Research is needed to identify more specific methods to triage women with low-grade squamous cervical lesions.     

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

Background  

Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells.     

Towards a computational method for imaging the extracellular potassium concentration during regional ischemia

We investigate the possibility of using body surface potential maps to image the extracellular potassium concentration during regional ischemia. The problem is formulated as an inverse problem based on a linear approximation of the bidomain model, where we minimize the difference between the results of the model and observations of body surface potentials. The minimization problem is solved by a one-shot technique, where the original PDE system, an adjoint problem, and the relation describing the minimum, are solved simultaneously. This formulation of the problem requires the solution of a 5 × 5 system of linear partial differential equations. The performance of the model is investigated by performing tests based on synthetic data. We find that the model will in many cases detect the correct position and approximate size of the ischemic regions, while some cases are more difficult to locate. It is observed that a simple post-processing of the results produces images that are qualitatively very similar to the true solution.     

RELATIONSHIP BETWEEN FISH AND THE NUMBER OF HORNS IN CERATIUM HIRUNDINELLA (DINOPHYCEAE): A FOOD‐WEB‐MEDIATED EFFECT ON ALGAL MORPHOLOGY?1

In a freshwater mesocosm experiment, we explored the potential for direct and indirect effects of roach (Rutilus rutilus) and Eurasian perch (Perca fluviatilis), two planktivorous fishes with different feeding behaviors, on the morphology of Ceratium hirundinella (O. F. Müll.) Dujard., a large dinoflagellate. Three morphs were detected: one with two hypothecal horns, one with a third rudimentary horn, and one with three well‐developed horns. We observed a strong negative relationship between the presence of fish and the proportion of three‐horned cells. The two fishes had strikingly similar effects on C. hirundinella morphology, despite their different capabilities to retain particles of the size of C. hirundinella. This finding suggests that the morphological variation in C. hirundinella was not related to selection by fish. Morphological variations in C. hirundinella could not be explained by fish‐mediated variations in turbidity (i.e., light climate) or by predation pressure by the fish. In contrast, the proportion of three‐horned cells was directly related to the biomass of filter‐feeding cladocerans. This result was unexpected since cladocerans are not considered to consume C. hirundinella and they did not depress C. hirundinella numbers in our experiment. Without excluding other possible mechanisms, we suggest that the third horn might help these dinoflagellates avoid physical contact with the filtering apparatus of the cladocerans and the consequent potential damage caused by these herbivores, which were more abundant in the absence of planktivorous fish.