Chance of live birth in the first pregnancy after referral among patients with recurrent pregnancy loss is not influenced by their relatives' reproductive history.

Purpose: Recurrent pregnancy loss (RPL) is defined as three or more consecutive pregnancy losses and affects 1-3% of couples trying to conceive. Pregnancy loss is more common among RPL patients' siblings than in the general population. Our objective was to investigate whether first-degree relatives with pregnancy losses influenced the chance of live birth in the first pregnancy after referral among women with RPL.Materials and methods: This is a cohort study of 2138 women with RPL seen at the Danish RPL Unit at Copenhagen University Hospital, Rigshospitalet between January 1st 2000 and December 31st 2017 with follow-up until December 2018. Pregnancies among first-degree relatives were reported by patients at their first consultation. Chance of live birth after referral was compared by logistic regression analysis.Results: Overall, 76% of the referred women achieved a pregnancy after referral and of these, 58% delivered a live born child. Women whose mother had experienced pregnancy loss were referred at a younger age than women with no pregnancy losses among first-degree relatives (mean age 33.6 (SD 4.6) versus 34.3 (SD 4.5), p = 0.002). Pregnancy losses among first-degree relatives did not influence chance of live birth.Conclusions: Our results indicate that pregnancy losses among first-degree family members is not an important risk factor for outcome of the first pregnancy after referral among women with RPL.

e-Vita: design of an innovative approach to COPD disease management in primary care through eHealth application.

BACKGROUND: COPD is a highly complex disease to manage as patients show great variation in symptoms and limitations in daily life. In the last decade self-management support of COPD has been introduced as an effective method to improve quality and efficiency of care, and to reduce healthcare costs. Despite the urge to change the organisation of health care and the potential of eHealth to support this, large-scale implementation in daily practice remains behind, especially in the Netherlands. METHODS/DESIGN: We designed a multilevel study, called e-Vita, to investigate different organisational implementation methods of a self-management web portal to support and empower patients with COPD in three different primary care settings. Using a parallel cohort design, the clinical effects of the web portal will be assessed using an interrupted times series (ITS) study design and measured according to changes in health status with the Clinical COPD Questionnaire (CCQ). The different implementations and net benefits of self-management through eHealth on clinical outcomes will be evaluated from human, organisational, and technical perspectives. DISCUSSION: To our knowledge this is the first study to combine different study designs that enable simultaneous investigation of clinical effects, as well as effects of different organisational implementation methods whilst controlling for confounding effects of the organisational characteristics. We hypothesize that an implementation with higher levels of personal assistance, and integrated in an existing care program will result in increased use of and satisfaction with the platform, thereby increasing health status and diminishing exacerbation and hospitalisation. TRIAL REGISTRATION: NTR4098 (31-07-2013).

e-Vita: design of an innovative approach to COPD disease management in primary care through eHealth application.

BACKGROUND: COPD is a highly complex disease to manage as patients show great variation in symptoms and limitations in daily life. In the last decade self-management support of COPD has been introduced as an effective method to improve quality and efficiency of care, and to reduce healthcare costs. Despite the urge to change the organisation of health care and the potential of eHealth to support this, large-scale implementation in daily practice remains behind, especially in the Netherlands. METHODS/DESIGN: We designed a multilevel study, called e-Vita, to investigate different organisational implementation methods of a self-management web portal to support and empower patients with COPD in three different primary care settings. Using a parallel cohort design, the clinical effects of the web portal will be assessed using an interrupted times series (ITS) study design and measured according to changes in health status with the Clinical COPD Questionnaire (CCQ). The different implementations and net benefits of self-management through eHealth on clinical outcomes will be evaluated from human, organisational, and technical perspectives. DISCUSSION: To our knowledge this is the first study to combine different study designs that enable simultaneous investigation of clinical effects, as well as effects of different organisational implementation methods whilst controlling for confounding effects of the organisational characteristics. We hypothesize that an implementation with higher levels of personal assistance, and integrated in an existing care program will result in increased use of and satisfaction with the platform, thereby increasing health status and diminishing exacerbation and hospitalisation. TRIAL REGISTRATION NUMBER: NTR4098 (31-07-2013).

Food in the cold: exploring food security and sovereignty in Whitehorse, Yukon.

Harsh weather patterns that are unpredictable owing to climate change, remoteness, dependence on food imports and limited local food production place Arctic and Subarctic food systems under serious pressure. The model of food sovereignty provides a surprisingly interesting contribution to address the food insecurity in these regions; it promotes long-term stable provision of healthy foods (sustainable) that are accessible to all (equity) and fosters local food production-consumption patterns (localisation). This study aims to deepen the understanding of food insecurity in the Subarctic regions and explores the possibilities for a food sovereignty approach at both individual and regional level. The study focuses on Whitehorse, capital of Yukon, Canada, and uses a cross-sectional online survey among residents of Whitehorse and semi-structured in-depth interviews with food-systems experts in Yukon. The findings indicated a need for affordable year-round local food production. Application of food sovereignty has provided the opportunities for local food procurement, innovation hubs, and several types of greenhouses including hydroponics and vertical farming, to work towards a more localised food system, thereby improving food security and sovereignty in Yukon. The findings constitute the scientific knowledge base for the formulation of prospective scenarios in the spirit of the food sovereignty theory.

Exposure-response analysis of allergy and respiratory symptoms in endotoxin-exposed adults.

The objective of the present study was to investigate exposure-response relationships between current endotoxin exposure and allergic and respiratory symptoms in adults, taking into account farming exposures during childhood. A cross-sectional study was conducted among 877 Dutch farmers and agricultural industry workers in 2006. Based on 249 full-shift personal airborne endotoxin samples, a job-exposure matrix was constructed to assign endotoxin exposure levels to all participants. Associations between endotoxin exposure and questionnaire data on symptoms were studied by multiple logistic regression. Adjusted odds ratios (OR) for an interquartile range increase in endotoxin level were elevated for respiratory symptoms such as wheezing (OR 1.41 (95% confidence interval 1.16-1.72)), wheezing with shortness of breath (1.50 (1.18-1.90)) and daily cough (1.29 (1.03-1.62)). In contrast, endotoxin was strongly associated with a decreased prevalence of hay fever (0.62 (0.49-0.78)). Workers who had grown up on a farm had a lower prevalence of hay fever, but no evidence was found of effect modification by farm childhood. In conclusion, occupational endotoxin exposure in adulthood is associated with an increased risk of asthma-like symptoms but a reduced prevalence of hay fever.

Feasibility and applicability of the paper and electronic COPD assessment test (CAT) and the clinical COPD questionnaire (CCQ) in primary care: a clinimetric study.

Three questionnaires are recommended in the management of chronic obstructive pulmonary disease by the global initiative for obstructive lung disease, of which two are the more comprehensive assessments: the chronic obstructive pulmonary disease assessment test and the clinical chronic obstructive pulmonary disease questionnaire. Both are carefully designed high-quality questionnaires, but information on the feasibility for routine use is scarce. The aim of this study was to compare the time to complete the chronic obstructive pulmonary disease assessment test and the clinical chronic obstructive pulmonary disease questionnaire and the acceptability of the questionnaires. Furthermore, the agreement between electronic and paper versions of the questionnaires was explored. The time to complete the electronic versions of the questionnaires was 99.6 [IQR 74; 157] vs. 97.5 [IQR 68; 136] seconds for clinical clinical chronic obstructive pulmonary disease questionnaire and chronic obstructive pulmonary disease assessment test, respectively. The difference in time to complete the questionnaire was not significant. The two questionnaires did not differ in "easiness to complete" or "importance of issues raised in questionnaires". Electronic vs. paper versions revealed high agreement (ICC CCQ = 0.815 [0.712; 0.883] and ICC CAT = 0.751 [0.608; 0.847]) between the administration methods. Based on this study it can be concluded that both questionnaires are equally suitable for use in routine clinical practice, because they are both quick to complete and have a good acceptability by the patient. Agreement between electronic and paper versions of the questionnaires was high, so use of electronic versions is justified.COPD: QUESTIONNAIRES EQUALLY SUITABLE FOR CLINICAL PRACTICE: Two questionnaires commonly used to manage chronic obstructive pulmonary diseases (COPD) are equally suitable for routine primary care. Researchers in The Netherlands, led by Janwillem Kocks from the University Medical Center Groningen, administered both the COPD assessment test (CAT) and the clinical COPD questionnaire (CCQ) to 95 patients with the lung disease. These two tests are the most comprehensive assessments recommended by the global initiative for obstructive lung disease for guiding treatment decisions. The researchers found that both tests took approximately 95-100 s on average. Both tests were also equally easy to complete and provided similar types of information. Most patients said they had no preference for either one, and they filled out both electronic and paper versions of the questionnaires in much the same way. The authors conclude that both tests seem fine for routine use.

Different characteristics of ferrochelatase in cultured fibroblasts of erythropoietic protoporphyria patients and normal controls.

Ferrochelatase activity was measured in crude extracts of fibroblasts, obtained from erythropoietic protoporphyria patients and healthy controls. The enzyme activity in erythropoietic protoporphyria fibroblasts was about 50% lower, compared to the controls. The sulfhydryl-oxidising reagent diamide inhibited the normal enzyme by about 50%, whereas ferrochelatase from erythropoietic protoporphyria fibroblasts was completely insensitive to the reagent. Pb2+ inhibits ferrochelatase activity by reacting with essential sulfhydryl groups. Low concentrations of Pb2+ inhibited the normal enzyme by 56%, but the mutant enzyme by only 8%. The photodynamic activity of bound mesoporphyrin substrate caused a biphasic inactivation of the normal enzyme. During the first 5 min of illumination a fast decrease of enzyme activity occurred to about 60% of the initial value. Experimental evidence indicates that this first phase of inactivation is caused by photooxidation of sulfhydryl groups. During further illumination inactivation continued at a much slower rate. With ferrochelatase from erythropoietic protoporphyria fibroblasts only the second, slow phase of photodynamic inactivation was observed. These observations suggest a mutation of ferrochelatase in erythropoietic protoporphyria, affecting the reactivity of sulfhydryl groups, involved in the catalytic activity of the enzyme.

Functional analysis of the human alpha 2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector.

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins.

The ability to couple genotype to phenotype has proven to be of immense value in systems such as phage display and has allowed genes encoding novel functions to be selected directly from complex libraries. However, the complexity of many eukaryotic proteins places a severe constraint on successful display in Escherichia coli. This restriction could be resolved if a eukaryotic virus could be similarly engineered for display purposes. Preliminary data have suggested that the baculovirus Autographa californica, a multiple nuclear polyhedrosis virus (AcMNPV) is a candidate for eukaryotic virus display because the insertion of peptides into the native virus coat protein, or the expression of foreign proteins as coat protein fusions, results in incorporation of the sequence of interest onto the surface of virus particles. A variety of strategies are currently under investigation to develop further the display capabilities of AcMNPV and to improve the complexity of library that might be accommodated. Several expression vectors for different forms of surface display have been developed and, coupled with improved recombination strategies, represent progress towards a refined tool for use in functional genomics and in vitro protein evolution.

Highly efficient production of GFP and its derivatives in insect cells for visual in vitro applications.

We have generated recombinant baculoviruses for expression of the green fluorescent protein (GFP), a bright GFP mutant (S65T), and a GFP-streptavidin fusion protein in Sf9 and High Five insect cell lines. At 3-4 days post infection, about 30% of the total protein contents was represented by the recombinant protein products, giving the infected insect cells a bright green color which was clearly visible by eye in daylight. The isolated GFP-streptavidin fusion protein, which possessed fluorescence properties identical to those of the native GFP, was capable of binding biotin as shown by using biotinylated beads as well as biotinylated antibody complexes decorating surface expressed GluR-6 glutamate receptor in live and fixed insect cells. The exceptionally high expression levels of GFP and GFP (S65T) and the GFP-streptavidin fusion protein in recombinant baculovirus infected insects should facilitate production of GFP derivatives for in vitro applications.

Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein.

A genetic fusion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fusion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (BEVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins from cells infected with the recombinant virus, VL1393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 micrograms/1 x 10(6) cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were functional with respect to their physical and enzymatic activities.

A baculovirus-expressed fusion protein containing the antibody-binding domain of protein A and insect luciferase.

A fusion construct encoding two antibody-binding sites of protein A from Staphylococcus aureus and click beetle, Pyrophorus plagiophthalamus, luciferase (LucGR) was designed and expressed using the baculovirus system. The construct was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in the insect Spodoperta frugiperda cell line during viral infection. The properties of the resultant chimeric protein product, protA-LucGR, were studied both in vivo and in vitro by using i) luminometry, ii) immunoblot analysis, iii) immunoprecipitation, iv) metabolic labeling procedures and v) luminescent immunoassays. Together, the results clearly demonstrate that the light-emitting properties of the fused luciferase construct remain intact. Further, the antibody-binding domain of protein A retains its activity as it binds to both rabbit and goat as well as human immunoglobulins. Due to the dual biological function of this fusion protein, it should provide a potential reagent within the field of molecular biology and diagnostics.

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells.

In order to study the processing of rubella virus (RV) structural proteins (capsid protein, of 33 kDa; E2 of 42-47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.

Flooding resistance and shoot elongation in relation to developmental stage and environmental conditions in Rumex maritimus L. and Rumex palustris Sm.

The flooding resistance of Rumex maritimus L. and Rumex palustris Sm., two species from frequently flooded areas of river forelands, was studied in two large-scale outdoor experiments. Plants were raised in cohorts and in several stages of development subjected to four weeks of submergence in early summer or in midsummer. The role of under-water shoot elongation in raising foliage above the water line was not equally important in both species. In R. maritimus, survival following either early- or midsummer flooding was restricted mainly to plants which emerged from the water surface by elongating their main shoot. R. palustris was better able to survive submergence, especially under early-summer conditions. As a consequence of survival being restricted to plants in the stem elongation stage, flooded plants of R. maritimus all reproduced in the first growing season and seed output correlated positively with stem length immediately after flooding. This was also the case for R. palustris flooded at midsummer. However, for plants of R. palustris which were flooded in early summer and remained below the water line, flowering was delayed or even postponed to the following season. Variation in seed weight was much larger in R. maritimus than in R. palustris and more clearly related to cohort and treatment effects. Differences between both species in Hooding resistance did not seem to be primarily caused by different abilities of shoot elongation, but by differences in submergence tolerance in relation to life-history characteristics.

The role of oxygen in submergence-induced petiole elongation in Rumex palustris: in situ measurements of oxygen in petioles of intact plants using micro-electrodes.

In a study on the mechanism of stimulated petiole elongation in submerged plants, oxygen concentrations in petioles of the flood-tolerant plant Rumex palustris were measured with micro-electrodes. Short-term submergence lowered petiole partial oxygen pressure to c. 19 kPa whereas prolonged submergence under continuous illumination depressed oxygen levels to c. 8-12 kPa after 24 h. Oxygen levels in petioles depended on the presence of the lamina, even in submerged conditions, and on available light. In darkness, petiole oxygen levels in submerged plants dropped quickly to values as low as 0.5-4 kPa. It is hypothesized that prolonged submergence in the light is accompanied by a decrease in carbon dioxide in the petiole. Submergence-enhanced petiolar elongation rate was compared with emergent plants. Peak daily elongation rates occurred at the end of the dark period in emergent plants, but in the middle of the light period in submerged plants. We suggest that this shift in daily elongation pattern is induced by dependence of growth on photosynthetically derived oxygen in submerged plants. Implications of reduced oxygen for ethylene production are raised. Levels of 1- aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase and ethylene sensitivity are cited as potential factors in hypoxia-induced ethylene release.

Flooding tolerance of Carex species in relation to field distribution and aerenchyma formation.

The flooding tolerance of Carex species was studied in relation to their field distribution and their capacity to form root aerenchyma under controlled conditions. In an alpine meadow, six Carex species were selected which were distributed in a clear zonation correlating with water content of the soil. Carex sempervirens and C. ferruginea were only found on nonflooded soil, the latter species preferring moister conditions. Carex davalliana and C. nigra were both associated with water-saturated soil, whereas C. limosa and C. rostrata preferred partially submerged conditions. Carex davalliana and C. limosa were bound to flooded soils with a relatively high redox potential and horizontally flowing groundwater. Carex rostrata and C. nigra grew in stagnant soil-flooded conditions with low soil redox potentials. The amount of aerenchyma in the roots of all species increased when grown in oxygen-deficient stagnant agar. This increase in root porosity, combined with increased root diameter, presumably improved internal aeration of the roots. Although all species survived experimental soil flooding, partial submergence was lethal to C. sempervirens and, surprisingly, also to the wetland species C. davalliana. Carex ferruginea showed a reduced growth rate during partial submergence. The three other species, all wetland plants, reached highest biomass production under soil-flooded and partially submerged conditions, with slower growth on free-draining soil. It is concluded that aerenchyma is not constitutive in the Carex species under study, and is best developed in Carex species from wetlands. Species with less aerenchyma perform poorly when soil-flooded, but conditions of partial submergence could even affect species with a considerable amount of root aerenchyma.

Root porosities and radial oxygen losses of Rumex and Plantago species as influenced by soil pore diameter and soil aeration.

The effects of hypoxia in ballotini and quartz sand with respectively large and small soil pore diameters on root porosity was studied for Rumex palustris Sra., Rumex acetosa L. and Plantago major L. ssp. major. Under hypoxic conditions R. palustris produced large root pores when large soil pores were present. Absence of such large soil pores resulted in the collapse of the root structure and a reduced root growth. Increases in intercellular spaces in P. major roots seemed to result in a higher root porosity upon hypoxia, a small soil pore diameter or a combination of both but this was not significant. Only a limited number of roots with large root pores was produced. The morphological structure of P. major roots without large root pores remained intact also in soils with a small soil pore diameter. R. acetosa only slightly increased its root porosity upon hypoxia and its morphological structure also remained intact when soil pore diameter was small. Radial oxygen loss (ROL) was found in R. palustris roots when grown in waterlogged, uncompacted soils. P. major had some roots with ROL in all treatments except in waterlogged, compacted soil. R. acetosa did not produce any roots showing ROL. Under field conditions aerenchyma is of no use in water saturated, compacted soils. This explains why a species like R. palustris only grows on hypoxic soil when it is not compacted, in contrast to P. major which will grow on moist, compacted soils. R. acetosa cannot be found on either very moist or heavily compacted soil, since it does not produce a healthy root system under these conditions.

Maturation of IgG avidity to individual rubella virus structural proteins.

BACKGROUND: the structural proteins of rubella virus, the capsid protein C and the envelope glycoproteins E1 and E2 were produced in lepidopteran insect cells using baculovirus expression vectors. The C-terminal ends of the corresponding proteins were fused to a polyhistidine tag for easy and gentle purification by metal ion affinity chromatography. OBJECTIVES: to investigate the maturation of natural and vaccinal IgG avidity against individual authentic and recombinant rubella virus (RV) structural proteins. STUDY DESIGN: the analysis was carried out using a modified immunoblotting technique where the purified baculovirus-expressed proteins were compared with authentic rubella virus proteins. Altogether, 47 well-characterised serum samples from both naturally infected patients and vaccines were studied. RESULTS: after natural RV infection, IgG antibodies specific for the E1 protein were predominant not only in terms of levels, but also in terms of rate and magnitude of avidity maturation. The avidity development of the IgG antibodies was much slower in vaccines than in patients after a natural RV infection. CONCLUSIONS: together, our results indicate that IgG avidity determination in conjunction with immunoblot analysis is useful in the diagnosis of a RV infection. The recombinant proteins showed similar reactivity patterns in the immunoblot analyses as compared with the authentic viral structural proteins, suggesting suitability for serodiagnostics.

Immunoblot analysis of natural and vaccine-induced IgG responses to rubella virus proteins expressed in insect cells.

BACKGROUND: The three structural proteins of rubella virus (RV), the capsid protein C and the envelope glycoproteins E1 and E2, were produced individually in soluble form in Sf9 insect cells using the baculovirus system. All proteins were equipped with a polyhistidine tag at their C-terminal ends to enable gentle purification by metal ion affinity chromatography. In addition, the E1 and E2 proteins were engineered to display the FLAG epitope tag at their N-terminal ends. STUDY DESIGN: The diagnostic potential of the recombinant purified proteins was evaluated by immunoblot and enzyme immuno assays (EIA) using a total of 57 well-characterised serum samples obtained at various time points after natural RV infection, congenital rubella syndrome (CRS), MMR vaccination or from controls with past RV immunity. In addition, acute and convalescent phase serum pools from a total of 20 patients were evaluated. Authentic RV proteins were used as a reference. RESULTS: The recombinant E1 and C proteins were predominant in eliciting the immune response in both postnatal and vaccinal RV infections, being much weaker in the vaccinal ones. The IgG response to the recombinant C protein was very strong after the first month post infection and decreased with time. The immune response against the recombinant E2 protein, however, was generally poor, but notably stronger after congenital infection. Together, the results showed that the individual recombinant protein antigens could be suitable for diagnosis of RV infection and for study of the immune response to rubella vaccination.