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Nurses are imperative for healthcare systems’ ability to effectively function during pandemics, yet multiple factors may affect their willingness and preparedness to work. The aim of the present cross-sectional survey was to identify factors associated with registered nurses’ (RN) willingness and preparedness to work during the novel coronavirus disease (COVID-19) pandemic. The study was reported following the STROBE guidelines. A total of 358 nurses completed a self-administered online questionnaire. The participants were a part of a COVID-19 task force at a Danish university hospital during spring 2020. The results showed that the majority of RNs felt a professional obligation to engage in clinical work during the pandemic; however, their willingness and preparedness to work were affected by multiple factors, such as being relocated voluntarily, being prepared for the task and feeling safe. This study highlights that these factors are essential for the hospital management and nurse leaders to take responsibility for. Nurses are imperative for healthcare systems’ ability to effectively function during pandemics, yet multiple factors may affect their willingness and preparedness to work. The aim of the present cross-sectional survey was to identify factors associated with registered nurses’ (RN) willingness and preparedness to work during the novel coronavirus disease (COVID-19) pandemic. The study was reported following the STROBE guidelines. A total of 358 nurses completed a self-administered online questionnaire. The participants were a part of a COVID-19 task force at a Danish university hospital during spring 2020. The results showed that the majority of RNs felt a professional obligation to engage in clinical work during the pandemic; however, their willingness and preparedness to work were affected by multiple factors, such as being relocated voluntarily, being prepared for the task and feeling safe. This study highlights that these factors are essential for the hospital management and nurse leaders to take responsibility for.

COVID-19 restrictions prevented relatives from visiting and accompanying patients to hospital and required that nurses wore personal protective equipment. These changes affected patients’ relationships with relatives and challenged their ability to connect with nurses. Individual, semi-structured interviews with 15 patients were carried out to explore patients’ experiences of their relationships with relatives and their collaboration with nurses during in- and outpatient contacts in non-COVID-19 hospital wards. The analysis of data was guided by phenomenological hermeneutic frame of reference and the study was reported according to the COREQ checklist. The findings illustrated that patients felt lonely and insecure when separated from relatives, caught between relatives and professionals during information exchange, and experienced the absence of relatives as both beneficial and burdening. Visitor restrictions provided patients with time to heal but prevented provision of informal care. Patients had to take responsibility for maintaining contact with relatives independent of their health condition. COVID-19 restrictions created distance with nurses, which potentially led to insufficient physical and psychosocial care.

To investigate the impact of combining the high-resolution (Hi-res) scan mode with deep learning image reconstruction (DLIR) algorithm in CT. Two phantoms (Catphan600® and Lungman, small, medium, large size) were CT scanned using combinations of Hi-res/standard mode and high-definition (HD)/standard kernels. Images were reconstructed with ASiR-V and three levels of DLIR. Spatial resolution, noise and contrast-to-noise ratio (CNR) were assessed. The radiation dose was recorded. The spatial resolution increased using Hi-res & HD. Image noise in the Catphan600® (69%) and the Lungman (10–70%) significantly increased when Hi-res & HD was applied. DLIR reduced the mean noise (54%). The CNR was reduced (64%) for Hi-res & HD. The radiation dose increased for both small (+70%) and medium (+43%) Lungman phantoms but decreased slightly for the large ones (−3%) when Hi-res was applied. In conclusion, the Hi-res scan mode improved the spatial resolution. The HD kernel significantly increased the image noise. DLIR improved the image noise and CNR and did not affect the spatial resolution.

The maintenance of physical distance, the absence of relatives and the relocation of registered nurses to COVID-19 units presumably affects nursing care at non-COVID-19 units. Using a qualitative design, this study explored registered nurses? experiences of how COVID-19 influenced nursing care in non-COVID-19 units at a Danish university hospital during the first wave of the virus. The study is reported using the COREQ checklist. The analysis offered two findings: (1) the challenge of an increased workload for registered nurses remaining in non-COVID-19 units and (2) the difficulty of navigating the contradictory needs for both closeness to and distance from patients. The study concluded that several factors challenged nursing care in non-COVID-19 units during the COVID-19 pandemic. These may have decreased the amount of contact between patients and registered nurses, which may have contributed to a task-oriented approach to nursing care, leading to missed nursing care. The maintenance of physical distance, the absence of relatives and the relocation of registered nurses to COVID-19 units presumably affects nursing care at non-COVID-19 units. Using a qualitative design, this study explored registered nurses’ experiences of how COVID-19 influenced nursing care in non-COVID-19 units at a Danish university hospital during the first wave of the virus. The study is reported using the COREQ checklist. The analysis offered two findings: (1) the challenge of an increased workload for registered nurses remaining in non-COVID-19 units and (2) the difficulty of navigating the contradictory needs for both closeness to and distance from patients. The study concluded that several factors challenged nursing care in non-COVID-19 units during the COVID-19 pandemic. These may have decreased the amount of contact between patients and registered nurses, which may have contributed to a task-oriented approach to nursing care, leading to missed nursing care.

To examine the diagnostic accuracy of spectral CT pulmonary angiography (S-CTPA) using ventilation-perfusions lung scintigraphy (V/Q-scan) as a reference standard in the diagnosis of acute or chronic pulmonary embolism (APE/CPE) and chronic thromboembolic pulmonary hypertension (CTEPH). PubMed, Embase, Scopus, and Web of Science were searched for the period from 1 Jan 2006 to 7 Feb 2019; eligible studies had > 10 patients over 18 years old, a diagnostic outcome of PE or CTEPH, and used V/Q scan as a reference standard. Bias and applicability were assessed using QUADAS-2 tools. Sensitivities, specificities, and predictive values were noted or calculated from available information. Meta-analysis employed a fixed-effects model of Mantel and Haenszel. Heterogeneity was assessed with I-squared statistics. Four hundred ninety-three unique records were identified. Following screening by title, 53 studies were included in the abstract and full-text assessment. A total of six articles were included; four were suitable for a meta-analysis. Pooled sensitivity was 94.2% (95% CI, 88.3–100%), pooled specificity was 88.5% (95% CI, 81.3–95.6%), and positive and negative predictive values were 87.8% (95% CI, 80.3–95.4%) and 94.5% (95% CI, 89.3–99.7%), respectively. Data on S-CTPA for PE/CTEPH remains promising, but limited; only small studies with methodological issues are available. Evidence is best for CPE/CTEPH whereas no firm conclusions are possible for APE. There is a need for larger, prospective studies with a robust composite reference standard including state-of-the-art CTPA and V/Q-scans. • S-CTPA has high sensitivity and specificity for perfusion defects in patients with PE or CPETH. • Methodological issues and diversity of reference standards were found in the small number of included studies. • There is a need for larger prospective studies with more robust composite reference standards.

The purpose was to examine if scatter correction software could replace a grid while maintaining image quality and reducing radiation dose for pelvic DR examinations. Grid images was produced with 70 kV and 16mAs. Anthropomorphic- and Contrast Detail RADiography (CDRAD) non-grid images were produced with 60 kV, 80 kV and 90 kV combined with five different mAs and scatter correction software. The anthropomorphic images were analyzed by absolute Visual Grading Analysis (VGA). The CDRAD images were analyzed using the CDRAD analysis software. The results showed a total of 54.6% non-grid images were evaluated as unsuitable for diagnostic use by the VGA. The CDRAD grid images showed that the IQF_inv values were significantly different (p = 0.0001) when compared to every group of non-grid images. Hereby, the conclusion stated that the scatter correction software did not compensate for the loss in image quality due to scattered radiation at the exposure levels included in a pelvic examination.

Aims The aim of this study was to evaluate the association between coronary computed tomography angiography (CCTA)-derived fractional flow reserve (FFRCT) and recurrent chest pain (CP) at 1-year follow-up in patients with stable angina pectoris (SAP). Methods and results Study of patients (n = 267) with SAP who underwent CCTA and FFRCT testing; 236 (88%) underwent invasive coronary angiography; and 87 (33%) were revascularized. Symptomatic status at 1-year follow-up was gathered by a structured interview. Three different FFRCT algorithms were applied using the following criteria for abnormality: (i) 2 cm-FFRCT ≤0.80; (ii) d-FFRCT ≤0.80; and (iii) a combination in which both a d-FFRCT ≤0.80 and a ΔFFRCT ≥0.06 must be present in the same vessel (c-FFRCT). Patients were classified into two groups based on the FFRCT test result and revascularization: completely revascularized/normal (CRN), patients in whom all coronary arteries with an abnormal FFRCT test result were revascularized or patients with completely normal FFRCT test results, and incompletely revascularized (IR), patients in whom ≥1 coronary artery with an abnormal FFRCT test result was not revascularized. Recurrent CP was present in 62 (23%) patients. Classification of patients (CRN or IR) was significantly associated with recurrent CP for all applied FFRCT interpretation algorithms. When applying the c-FFRCT algorithm, the association with recurrent CP was found, irrespective of the extent of coronary calcification and the degree of coronary stenosis. A negative association between per-patient minimal d-FFRCT and recurrent CP was demonstrated, P < 0.005. Conclusion An abnormal FFRCT test result is associated with an increased risk of recurrent CP in patients with new-onset SAP.

The purpose of this study was to investigate the challenges in comparing digital radiography (DR) systems from different vendors for various combinations of exposure factors in posterior–anterior hand radiographs. Image quality was evaluated for a range of tube voltages and tube current-time products using a technical contrast-detail (CDRAD) phantom and an anthropomorphic hand phantom. 900 technical CDRAD images were analysed providing quality figures of merit (IQFinv) and two experienced reporting radiographers using visual grading analysis (VGA) scored 108 anthropomorphic images. This study demonstrates the differences between the DR systems included. When compensating for variations in dose, Canon showed superior results for technical image quality and Fuji for visual image quality for a standard dose point at DR hand examination (ln(DAP) 1.1, 50 kV and 2.5 mAs).

Baggrund: Evidensbaseret praksis er væsentlig med henblik på at udøve sygepleje af høj kvalitet og sikre, at den enkelte patients behov imødekommes. Evidensbaseret praksis kræver bl.a. udvikling af sygeplejerskers forskningsmæssige kompetencer. Studier peger på, at sygeplejerskers forskningsmæssige kompetencer kan forbedres gennem en struktureret indsats. Formålet var at undersøge, om et kombineret projekt-, vejlednings- og undervisningsforløb har betydning for sygeplejerskers vurdering af egne forskningsmæssige kompetencer. Metode: En spørgeskemaundersøgelse som blev sendt til de deltagende sygeplejersker med målinger før og efter et kombineret projekt-, vejlednings- og undervisningsforløb. Kursusforløbet for kliniske sygeplejersker indeholdt en workshop efterfulgt af otte læringsmoduler indeholdende 11 undervisningsdage fordelt over to år. Resultater: Sygeplejerskerne har udviklet deres forskningskompetencer. Ved kursusforløbets start svarede 58,6%, at de kun havde begrænsede eller ingen forskningsmæssige færdigheder, hvorimod dette gjorde sig gældende for 4,8% på kursusforløbet sidste dag. Specielt synes der at være sket forbedringer relateret til forståelse og varetagelse af såvel kvantitative som kvalitative forskningsdesigns samt formulering og udvikling af forskningsspørgsmål og forskningsdesign. Konklusion: Sygeplejerskerne har forbedret deres forskningskompetencer markant. Projekt-, vejlednings- og undervisningsforløbet har potentiale til at styrke linket mellem forskning og klinisk praksis. Kursusforløbet vil med fordel kunne udbredes til andre hospitaler og institutioner, der har et ønske om eller interesse i at øge forskningsaktiviteter og forskningskompetencer blandt kliniske sygeplejersker, opbygge forskningskapacitet og styrke evidensbaseret praksis i klinisk sygepleje. Background: Evidence-based practice is imperative to ensure high quality nursing care and to meet the patients’ needs. Evidence-based practice requires a development of nurses’ research competencies. Studies show that a structured education program can support the development of nurses’ research competencies. The aim of this study was to examine the influence of a combined project-, supervision and education program on nurses’ self-reported research competencies. Methods: A questionnaire was conducted before and after a combined project-, supervision and education program. It was distributed to all participating nurses. The program consisted of a 2-year course for clinical nurses encompassing a workshop followed by eight modules and 11 days of education. Results: Nurses participating in the program developed their research competencies. Prior to participating, 58.6% of the nurses answered that they had limited or none skills within research whereas this was only the case for 4.8 % following participation in the program. In particular, improvements relate to understanding and handling quantitative and qualitative research designs as well as formulating and developing research questions and research designs. Conclusion: Nurses significantly improved their research competencies. The project-, supervision and education program has the potential to strengthen the link between research and clinical practice and be disseminated to other hospitals and institutions who wish to increase research activities, improve research competencies among clinical nurses and build research capacity within the institution. Furthermore, it may have the potential to strengthen evidence-based practice in clinical nursing.

Objectives: Shiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea, and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal samples of patients with suspected infective gastroenteritis were analysed for STEC. Methods: In this population-based cohort study, all stool samples referred to two clinical microbiology laboratories were screened for STEC by culture and/or PCR. Epidemiological (n=170) and clinical (n=209) characteristics were analysed using data from local and national registries. Results: Overall, 75,132 samples from 30,073 patients were screened resulting in 217 unique STEC-isolates. The epidemiological analysis showed an incidence of 10.1 cases per 100,000 person-years, which was more than twofold higher than the incidence in the rest of Denmark (3.4 cases per 100,000 person-years, p 1-only isolates) showed a lower frequency of acute (n=11, p 1-only isolates and long-term gastrointestinal symptoms.

The hypomyelinating leukodystrophies (HMLs) encompass the X-linked Pelizaeus-Merzbacher disease (PMD) caused by PLP1 mutations and known as the classical form of HML as well as Pelizaeus-Merzbacher-like disease (PMLD) (Online Mendelian Inheritance in Man [OMIM] 608804 and OMIM 260600) due to GJC2 mutations. In addition, mutations in at least 10 other genes are known to cause HMLs. In 2008, an Israeli family with clinical and neuroimaging findings similar to those found in PMD was reported. The patients were found to have a homozygous missense mutation in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60), and the disorder was defined as the autosomal recessive mitochondrial Hsp60 chaperonopathy (MitCHAP-60) disease. We here report the first case of this severe neurodegenerative disease since it was first described. Given the fact that the families carried the same mutation our patient probably belongs to the same extended family as the Israeli family. In conclusion, the MitCHAP-60 disease should be considered as a rare differential diagnosis in HML.

REVIEW OBJECTIVE: The objective is to identify the best available evidence on the effectiveness of primary nursing in the care and satisfaction of adult inpatients.

Background: Oxygen is liberally administered in intensive care units (ICUs). Nevertheless, ICU doctors’ preferences for supplementing oxygen are inadequately described. The aim was to identify ICU doctors’ preferences for arterial oxygenation levels in mechanically ventilated adult ICU patients. Methods: In April to August 2016, an online multiple-choice 17-part-questionnaire was distributed to 1080 ICU doctors in seven Northern European countries. Repeated reminder e-mails were sent. The study ended in October 2016. Results: The response rate was 63%. When evaluating oxygenation 52% of respondents rated arterial oxygen tension (PaO2) the most important parameter; 24% a combination of PaO2 and arterial oxygen saturation (SaO2); and 23% preferred SaO2. Increasing, decreasing or not changing a default fraction of inspired oxygen of 0.50 showed preferences for a PaO2 around 8 kPa in patients with chronic obstructive pulmonary disease, a PaO2 around 10 kPa in patients with healthy lungs, acute respiratory distress syndrome or sepsis, and a PaO2 around 12 kPa in patients with cardiac or cerebral ischaemia. Eighty per cent would accept a PaO2 of 8 kPa or lower and 77% would accept a PaO2 of 12 kPa or higher in a clinical trial of oxygenation targets. Conclusion: Intensive care unit doctors preferred PaO2 to SaO2 in monitoring oxygen treatment when peripheral oxygen saturation was not included in the question. The identification of PaO2 as the preferred target and the thorough clarification of preferences are important when ascertaining optimal oxygenation targets. In particular when designing future clinical trials of higher vs lower oxygenation targets in ICU patients.

AIMS AND OBJECTIVES: To describe the incidence of problems associated with the positioning of anaesthetised surgical patients.BACKGROUND: The positioning of the anaesthetised surgical patient is a complex task. The interdisciplinary nature with several professional groups in a surgical team may lead to conflict between the positioning standards and individual consideration for the patient. Existing knowledge of the relationship between the different positioning forms, surgical team competences and the applicability and availability of positioning equipment is sparse.DESIGN: A descriptive cross-sectional study.METHOD: An electronic questionnaire was sent to 833 OR nurses employed at four public university hospitals. With 481 responses, a response rate of 57·7% was achieved. Descriptive statistical analyses were performed using the spss software package (version 19.00).RESULTS: Positioning of the patient was found to be particularly difficult for the prone (43·8%), lithotomy (53·4%) and lateral (65·5%) positions. Lack of positioning competences and equipment for arm support, standardised equipment for leg support and standard sizes of OR beds seemed to complicate positioning.CONCLUSION: Lack of appropriate positioning equipment and positioning competences in surgical teams, combined with the poor availability of positioning equipment in ORs were found to cause problems.RELEVANCE TO CLINICAL PRACTICE: There is a need for innovative solutions to develop modern forms of positioning equipment allowing individual consideration of the patient. Further research is required on positioning equipment, optimisation of continuity and the establishment of permanent surgical teams.

REVIEW QUESTION/OBJECTIVE The objective of this review is to identify and synthesize the best available knowledge on how community-dwelling frail elderly people experience daily life and what they find meaningful in daily life. Specifically the review questions are: • How do community-dwelling frail elderly people experience daily life? • What do community-dwelling frail elderly people find meaningful in daily life? INCLUSION CRITERIA Types of participants: The review will consider studies that include frail elderly community-dwelling people, aged 65 years or more, of either gender. For the purpose of this review the definition of frailty will be based on a multidimensional integrated approach including the physical, psychological and social aspects of frailty. Studies will be included in the systematic review if inclusion criteria such as a screening tool for frailty or known risk factors for frailty are used to identify and describe the participants, who were included in the studies. Known risk factors are, amongst others, being aged 75 years or more, comorbidity, living alone, low income and low educational levels. Phenomena of interest: This review will consider studies investigating the experiences of daily life for community-dwelling frail elderly and the themes they find meaningful in daily life. Context: This review will consider studies involving community-dwelling frail elderly people, living alone or with a spouse in their own home.

The early-life stages of development of the calanoid copepod Acartia tonsa from egg to copepodite I is proposed as an endpoint for assessing sediment toxicity by exposing newly released eggs directly onto the sediment-water interface. A preliminary study of 5 sediment samples collected in the lagoon of Venice highlighted that the larval development rate (LDR) and the early-life stages (ELS) mortality endpoints with A. tonsa are more sensitive than the standard amphipod mortality test; moreover LDR resulted in a more reliable endpoint than ELS mortality, due to the interference of the sediment with the recovery of unhatched eggs and dead larvae. The LDR data collected in a definitive study of 48 sediment samples from the Venice Lagoon has been analysed together with the preliminary data to evaluate the statistical performances of the bioassay (among replicate variance and minimum significant difference between samples and control) and to investigate the possible correlation with sediment chemistry and physical properties. The results showed that statistical performances of the LDR test with A. tonsa correspond with the outcomes of other tests applied to the sediment-water interface (Strongylocentrotus purpuratus embryotoxicity test), sediments (Neanthes arenaceodentata survival and growth test) and porewater (S. purpuratus); the LDR endpoint did, however, show a slightly higher variance as compared with other tests used in the Lagoon of Venice, such as 10-d amphipod lethality test and larval development with sea urchin and bivalves embryos. Sediment toxicity data highlighted the high sensitivity and the clear ability of the larval development to discriminate among sediments characterized by different levels of contamination. The data of the definitive study evidenced that inhibition of the larval development was not affected by grain-size and the organic carbon content of the sediment; in contrast, a strong correlation between inhibition of the larval development and the sediment concentrations of some metals (Cu, Hg, Pb, Zn), acid-volatile sulphides (AVS), polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs) was found. No correlation was found with DDTs, hexachlorobenzene and organotin compounds.

The antimicrobial sulfonamide sulfadiazine has in the last decades been detected in environmental water bodies, both surface and ground water. Since pH in the environment may vary considerably, this study examined the toxicity of the amphoter sulfadiazine towards Daphnia magna at pH levels of 6.0, 7.5 and 8.5, thus taking the impact of speciation into consideration, contrary to earlier eco-toxicity studies conducted at standard conditions. Toxicity tests were performed using the standard ISO 6341 test procedure modified to accommodate the three pH levels and the toxicity was expressed as EC50. After 48 h the EC50 was determined to be 27.2, 188 and 310 mg L(-1) at pH 6.0, 7.5 and 8.5, respectively, thus demonstrating a significant effect of pH on the toxicity of sulfadiazine. Furthermore, the bioconcentration factor (dry weight) was determined to be 50 and 36 at pH 6.0 and 8.5, respectively. The higher toxicity at the lower pHs was assumed to be caused by the higher fraction of un-ionized sulfadiazine at the lower pHs. However, the one and a half fold higher bioconcentration at pH 6.0 relative to pH 8.5 does not match the more than ten times higher toxicity at pH 6.0. When comparing the fraction of neutral compound to toxicity and bioconcentration results neither toxicity nor bioconcentration can be ascribed solely to the unionized fraction of sulfadiazine.

The exposure of non-target aquatic organisms to pesticides often happens as short-term, high exposure events (pulses) and effects of these must be addressed in the current regulation in the EU. It is, however, questionable whether the effects of pulse exposures are adequately covered by the standardized ecotoxicological tests used in environmental effect assessments, since these aim at maintaining constant exposure concentrations during the incubation. Therefore, we investigated the effects of four sulfonylurea herbicides (flupyrsulfuron-methyl, metsulfuron-methyl, rimsulfuron, and thifensulfuron-methyl) on the growth of Lemna gibba over a 6-day period after 24h of pulse exposure, and compared with effects observed in standard OECD tests with continuous exposure. It was observed that concentrations around the E(y)C50-values found in OECD tests did not affect the growth in the 6 days post-exposure period. Slightly higher concentrations initially resulted in lower growth in pulse exposure tests, but the growth rate of the plants reached the level of untreated plants during the 6 days post-exposure period. The 24h pulse exposure tests gave 2-6 times higher E(y)C50-values than the OECD 7-d continuous exposure tests. The approach of this study enables experimentally based comparisons between observations of effects between the two exposure regimes. We propose that results obtained in this way be applied in effect assessments for intermittent releases.

The study was performed to detect the effects of anti-androgenic compounds on the reproduction. In this paper alterations observed in the marine calanoid copepod Acartia tonsa exposed to environmental concentrations of cyproterone acetate (CPA), linuron (LIN), vinclozolin (VIN), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) for 21 days covering a full life cycle are described. Histological alterations were studied with a focus on reproductive organs, gonad and accessory sexual glands. Exposure to ≥1.2 µg L(-1) CPA caused degeneration of spermatocytes and deformation of the spermatophore in males. In a single male exposed to 33 µg L(-1) CPA, an ovotestis was observed. In CPA exposed females, enhancement of oogenesis, increase in apoptosis and a decrease in proliferation occurred. Exposure of males to ≥12 µg L(-1) LIN caused degenerative effects in spermatogonia, spermatocytes and spermatids, and at 4.7 µg L(-1) LIN, the spermatophore wall displayed an irregular formation. In LIN exposed females, no such structural alterations were found; however, the proliferation index was reduced at 29 µg L(-1) LIN. At an exposure concentration of ≥100 µg L(-1) VIN, distinct areas in male gonad were stimulated, whereas others displayed a disturbed spermatogenesis and a deformed spermatophore wall. In VIN exposed female A. tonsa, no effects were observed. Male A. tonsa exposed to p,p'-DDE displayed an impairment of spermatogenesis in all stages with increased degrees of apoptosis. In p,p'-DDE-exposed females, a statistical significant increase of the proliferation index and an intensification of oogenesis were observed at 0.0088 µg L(-1).

Object The authors describe the first clinical trial with encapsulated cell biodelivery (ECB) implants that deliver nerve growth factor (NGF) to the cholinergic basal forebrain with the intention of halting the degeneration of cholinergic neurons and the associated cognitive decline in patients with Alzheimer disease (AD). The NsG0202 implant (NsGene A/S) consists of an NGF-producing, genetically engineered human cell line encapsulated behind a semipermeable hollow fiber membrane that allows the influx of nutrients and the efflux of NGF. The centimeter-long capsule is attached to an inert polymer tether that is used to guide the capsule to the target via stereotactic techniques and is anchored to the skull at the bur hole. Methods Six patients with mild to moderate AD were included in this Phase Ib open-label safety study and were divided into 2 dose cohorts. The first cohort of 3 patients received single implants targeting the basal nucleus of Meynert (Ch4 region) bilaterally (2 implants per patient), and after a safety evaluation, a second cohort of 3 patients received bilateral implants (a total of 4 implants per patient) targeting both the Ch4 region and the vertical limb of the diagonal band of Broca (Ch2 region). Stereotactic implantation of the devices was successfully accomplished in all patients. Despite extensive brain atrophy, all targets could be reached without traversing sulci, the insula, or lateral ventricles. Results Postoperative CT scans allowed visualization of the barium-impregnated tethers, and fusion of the scans with stereotactic MR images scan was used to verify the intended positions of the implants. Follow-up MRI at 3 and 12 months postimplantation showed no evidence of inflammation or device displacement. At 12 months, implants were successfully retrieved, and low but persistent NGF secretion was detected in half of the patients. Conclusions With refinement, the ECB technology is positioned to become an important therapeutic platform in restorative neurosurgery and, in combination with other therapeutic factors, may be relevant for the treatment of a variety of neurological disorders. Clinical trial registration no.: NCT01163825.

Self-tracking technologies, wearables and the so-called idea of the quantified self are gaining increasing acceptance within organizational life. These technologies enable new territories for manag...

Based on a literature review and an overview of toxic effects of engineered nanoparticles in aquatic invertebrates, this paper proposes a number of recommendations for the developing field of nanoecotoxicology by highlighting the importance of invertebrates as sensitive and relevant test organisms. Results show that there is a pronounced lack of data in this field (less than 20 peer-reviewed papers are published so far), and the most frequently tested engineered nanoparticles in invertebrate tests are C(60), carbon nanotubes, and titanium dioxide. In addition, the majority of the studies have used Daphnia magna as the test organism. To date, the limited number of studies has indicated acute toxicity in the low mg l(-1) range and higher of engineered nanoparticles to aquatic invertebrates, although some indications of chronic toxicity and behavioral changes have also been described at concentrations in the high microg l(-1) range. Nanoparticles have also been found to act as contaminant carriers of co-existing contaminants and this interaction has altered the toxicity of specific chemicals towards D. magna. We recommend that invertebrate testing is used to advance the level of knowledge in nanoecotoxicology through standardized short-term (lethality) tests with invertebrates as a basis for investigating behaviour and bioavailability of engineered nanoparticles in the aquatic environment. Based on this literature review, we further recommend that research is directed towards invertebrate tests employing long-term low exposure with chronic endpoints along with more research in bioaccumulation of engineered nanoparticles in aquatic invertebrates.

Acute and reproductive toxicity tests were conducted on seven strains of Daphnia magna from six laboratories in five countries. 3,4-Dichloroaniline (DCA) and fenoxycarb were used as test chemicals. Acute toxicity tests revealed that estimated EC(50) (50% effective concentration) values for DCA varied by a factor of 2.1 among strains (310-640 microg/L), whereas the EC(50) values for fenoxycarb varied by a factor of 4 (210-860 microg/L). EC(50) values for reproductive toxicity tests with DCA ranged from 5.9 to 38 microg/L among strains. Fenoxycarb exposure induced the production of male neonates in all the strains used in the present study. Estimated EC(50) values for the induction of male offspring were highly variable among strains: sensitivity to fenoxycarb differed by a factor of approximately 23 overall (0.45-10 microg/L). The present pre-validation tests suggest that induction of male sex in neonates by a juvenile hormone analog is universal among genetically different strains. Decreased total numbers of neonates at increased concentrations of fenoxycarb as well as other juvenoids may, however, obscure the incidence of male neonates production in the 21-day reproduction tests due to the low statistical power.

New and updated methods to detect and characterize endocrine disrupting chemicals (EDCs) are urgently needed for the purpose of environmental risk assessment since these substances are often not detected using existing chronic toxicity tests. Numerous reports on the effects of EDCs on crustacean development and reproduction have been published and the development of life-cycle tests with crustaceans has been prioritized within the OECD work program for endocrine disrupter testing and assessment. As a result, Sweden, and Denmark initiated a proposal for development of a full life-cycle test with marine copepods (Acartia tonsa, Nitocra spinipes, Tisbe battagliai, and Amphiascus tenuiremis). The present paper gives an overview on the endocrine system of crustaceans with special emphasis on development and reproduction, which are targets for endocrine disruption, and reviews available methods for detecting effects on development and reproduction in calanoid and harpacticoid copepods. A draft OECD guideline Copepod Development and Reproduction Test has been developed, and a pre-validation of this draft guideline was completed in 2005. An updated draft guideline, taking into account the results of the pre-validation, is now under validation in an international ring-test, which is running till the end of 2006.

Objective – To assess the presence of Epstein–Barr virus (EBV) and human herpesvirus 6B (HHV-6B) DNA in saliva and plasma from multiple sclerosis (MS) patients enrolled in a randomized, double-blind, placebo-controlled valacyclovir treatment study. Methods – DNA was prepared following ultracentrifugation of saliva and plasma. EBV and HHV-6B DNAs were determined by real-time polymerase chain reaction. Results – EBV and HHV-6B DNAs were detected in 41% and 65% of saliva samples, respectively. In patients treated with valacyclovir, the percentage of saliva samples with EBV was significantly reduced (9%; P = 0.000017), whereas the frequency of HHV-6B positive samples was unchanged (57%; P = 0.38). Longitudinal studies demonstrated a time-dependent reduction in the frequency of saliva samples containing EBV following valacyclovir treatment. In contrast, plasma contained EBV and HHV-6B DNAs in 17% and 25% of the samples, respectively, and these numbers were not significantly reduced following valacylovir treatment (13% and 16%, respectively), nor were they different from those of healthy controls (6% and 39%, respectively). Patients with high disease activity had a significantly higher frequency of EBV (P = 0.018) and HHV-6B (P = 0.023) positive samples than did patients with low disease activity. The presence of EBV and HHV-6B was strongly correlated in plasma (P

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca2+, Mn2+, Mg2+, Ba2+, Li2+, NaN3 and isopropanol was observed. The K m and V max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.

Background Generation of multiplanar reformation (MPR) images has become automatic on most modern computed tomography (CT) scanners, potentially increasing the workload of the reporting radiologists. It is not always clear if this increases diagnostic performance in all clinical tasks. Purpose To assess detection performance using only coronal multiplanar reformations (MPR) when triaging patients for lung malignancies with CT compared to images in three orthogonal planes, and to evaluate performance comparison of novice and experienced readers. Material and Methods Retrospective study of 63 patients with suspicion of lung cancer, scanned on 64-slice multidetector computed tomography (MDCT) with images reconstructed in three planes. Coronal images were presented to four readers, two novice and two experienced. Readers decided whether the patients were suspicious for malignant disease, and indicated their confidence on a five-point scale. Sensitivity and specificity on per-patient basis was calculated with regards to a reference standard of histological diagnosis, and compared with the original report using McNemar’s test. Receiver operating characteristic (ROC) curves were plotted to compare the performance of the four readers, using the area under the curve (AUC) as figure of merit. Results No statistically significant difference of sensitivity and specificity was found for any of the readers when compared to the original reports. ROC analysis yielded AUCs in the range of 0.92–0.93 for all readers with no significant difference. Inter-rater agreement was substantial (kappa = 0.72). Conclusion Sensitivity and specificity were comparable to diagnosis using images in three planes. No significant difference was found between experienced and novice readers.

Battery tests serve as integral tools to decide whether a treatment process is ecotoxicologically safe or not. In the present study, a battery of toxicity tests was employed to elucidate the toxicity of the potential endocrine-disrupting pollutant bisphenol A (BPA) and its advanced oxidation products. For this purpose, BPA was subjected to Fenton treatment in the growth medium of the test organisms employed as well as in real lake water. Treatment results indicated that BPA removals were fast and complete within less than a minute, whereas total organic carbon (TOC) removals were rather incomplete, speaking for the accumulation of refractory degradation products. The presence of chloride and/or natural organic matter influenced H2O2 consumption rates and the treatment performance of the Fenton’s reagent as well. The sensitivity of the selected test organisms for BPA and its Fenton treatment products in different water matrices was found in the following decreasing order: the freshwater microalgae (Pseudokirchneriella subcapitata) > the freshwater cladoceran (Daphnia magna) > marine photobacteria (Vibrio fischeri).

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X 1 ), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX 1 X 2 GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 °C or 37 °C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 °C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNA f Met binding.

The acute and chronic toxicity of nine antibiotics used both therapeutically and as growth promoters in intensive farming was investigated on the freshwater crustacean Daphnia magna. The effect of the antibiotics metronidazole (M), olaquindox (OL), oxolinic acid (OA), oxytetracycline (OTC), streptomycin (ST), sulfadiazine (SU), tetracycline (TC), tiamulin (TI) and tylosin (TY) was tested in accordance to the ISO (1989) and OECD (1996) standard procedures. The acute toxicities (48-h EC50 value, mg/l) in decreasing order were OA (4.6), TI (40), SU (221), ST (487), TY (680) and OTC (approximately 1000). NOECs were 340 mg/l for TC and 1000 mg/l for M and OL. Toxic effect on reproduction occurred generally at concentrations, which were one order of magnitude below the acute toxic levels. The chronic toxicity (EC50 values, mg/l) in the D. magna reproduction test in decreasing order were TI (5.4), SU (13.7), TC (44.8) and OTC (46.2). The NOECs (mg/l) obtained in the reproduction test with OA, ST, TY and M were 0.38 for OA, 32 for ST, 45 for TY and 250 for M. The observed toxicity of OA to D. magna indicates that this substance, which is a commonly used feed additive in fish farms, has a potential to cause adverse effects on the aquatic environment.

Changes in algal nitrogen status that increase algal lipid content also affect the bioconcentration of hydrophobic organic compounds (HOCs). Bioconcentration factors (BCFs) for several HOCs increased up to nine times as the total algal lipid content of the green algae Selenastrum carpricornutum increased from 17 to 44% of the algal dry weight as a consequence of nitrogen starvation. An increase in total lipid from 17 to 44% should theoretically increase the BCFs by a factor of 2.6. BCFs for PCB 31, PCB 49, PCB 153, and DDT increased with maximum lipid content by factors of 6.3, 8.9, 8.9, and 6.6, respectively, thus more than theoretically predicted from the lipid normalization of BCFs obtained at exponential growth phase (17% total lipid for S. carpricornutum), whereas BCFs for PCB 105, phenanthrene, and 4-chloroaniline increased at 44% lipid content, only by factors of 1.5, 1.5, and 2.5, respectively, and thus less than or equal to the theoretical prediction. Lipid-class normalization of BCFs did not reveal significant information beyond that available from normalizing to total lipid.

Literature on the basic endocrinology of crustaceans, such as crab, lobster and shrimp, suggest that estrogens, e.g., 17beta-estradiol, are stimulating hormones in female sexual maturation and egg production in crustaceans. The copepod Acartia tonsa, has continuous egg production. A profile of the egg production per time unit through the maturation of this species may be used as an expression of the degree of maturation of the female internal reproductive organs. Comparison of control profiles with profiles of estrogen-exposed copepods may thus demonstrate that estrogen exposure results in a steeper maturation profile. The proposed endpoint was identified by exposing copepods to the natural estrogen 17beta-estradiol and the antropogenic estrogen bisphenol A. Both compounds produced significant effects at 23 and 20 microg/L, respectively. Since bisphenol A is traditionally believed to be less estrogenic than 17beta-estradiol, the nonestrogen 2, 3-dichlorophenole was tested at 13.6 microg/L (molar concentration equal to 23 microg/L 17beta-estradiol) to test if the response is a general nonestrogenic toxic stress effect. The 2,3-dichlorophenole produced no effect.

Polypeptide chain termination in Escherchia coli is known to require two codon specific release factors. RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract: a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.

Ribosome release factor (RRF) from Escherichia coli was overproduced from an osmo-expression vector. More than 40% of cell protein was RRF after 6 h of induction. A purification scheme is described that produced 50 mg of RRF from an initial culture of 2 L. The recycling time for ribosomes synthesising the tripeptide fMet-Phe-Leu in vitro in the absence of RF3 was reduced from 40 to 15 s by the addition of purified 1.5 μM RRF.

The marine calanoid copepod Acartia tonsa was exposed to methyltestosterone (MET, 1.6-126 μg L(-1)), letrozole (LET, 10-1000 μg L(-1)), triphenyltin chloride (TPT, 0.0014-0.0088 μg L(-1) TPT-Sn) and fenarimol (FEN, 2.8-105 μg L(-1)) for 21 d covering a full life-cycle. All four compounds investigated are known to act as androgens in vertebrates. The digestive tract, musculature, nervous system, reproductive organs, gonad and accessory sexual glands were examined by light microscopy after routine staining and immune-labelling for detection of apoptosis and determination of proliferation activities. MET induced an inhibition of oogenesis, oocyte maturation and yolk formation, respectively, which was most pronounced at the lowest concentrations tested. In LET exposed males, spermatogenesis was enhanced with very prominent gamete stages; in some stages apoptosis occurred. The spermatophore was hypertrophied and displayed deformations. In females, LET induced a disorder of oogenesis and disturbances in yolk synthesis. TPT stimulated the male reproductive system at 0.0014 and 0.0035 μg TPT-SnL(-1), whereas inhibiting effects were observed in the female gonad at 0.0088 μg TPT-SnL(-1). In FEN exposed females proliferation of gametes was reduced and yolk formation showed irregular features at 2.8-105 μgL(-1). In FEN exposed males an elevated proliferation activity was observed. No pathological alterations in other organ systems, e.g. the digestive tract including the hindgut acting as respiratory organ, the nervous system, or the musculature were seen. This indicates that the effects on gonads might be caused rather by disturbance of endocrine signalling or interference with hormone metabolism than by general toxicity.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

The toxic effects of potassium dichromate (K 2 Cr 2 O 7 ), 3,4-dichloroaniline (DCA) and 2,4-dichlorophenol (DCP) on the photosynthesis of natural marine phytoplankton and five species of marine microalgae were investigated. Effect concentrations corresponding to a 50 % depression of photosynthesis (6h EC 50 ) were found within relatively narrow ranges of 3.3–7.2 mg/l and 1.5–12 mg/l for DCA and DCP respectively, whereas the sensitivities towards K 2 Cr 2 O 7 varied much more. The effects of DCA and DCP on the growth rate of some species were also investigated. EC 50 values for DCP and DCA were found within the ranges 0.5–20 mg/l and 1.1–2.6 mg/l respectively. Diatoms were generally found to be the most sensitive species.

For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system. The gen infB was cloned into the thermo-inducible runaway plasmid pCP40 [1] and subsequently transformed into the E coli strain C600[pc1857]. In this system the expression of infB is under the control of the strong promoter λ P l and the cells carry the plasmid pcI857, which contains a thermosensible λcI repressor. Overexpression of IF2, which is approximately 30 times higher than the expression in wild-type-cells, in induced at 42°C and continues for 2 h at 37°C. From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS. In approximately 8 h, 5 mg of pure and active IF2 can be obtained from 10 g overproducing cells. This corresponds to 5 mg of IF2 per litre of medium. The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNA f Met to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA. Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates. In addition to the higher flow capacity of Q-sepharose HP, this new matrix can be loaded with an S30 supernatant. This avoids the need for the preparation of a ribosome-free S100 supernatant by ultracentrifugation and thus the need to keep IF2 in solution with membrane and periplasmatic proteases for at least 2–3 h. By this procedure, only minor amounts of the proteolytic 65 kDA fragment IF2γ are formed during the purification of IF2. Stability tests showed that IF2 is extremely labile in the S30 supernatant. However, the purified IF2 is not an unstable protein as previously believed.

A suggested standard test protocol for the short term14C assimilation algal toxicity test method (photosynthesis inhibition test) has been evaluated with natural phytoplankton and cultures of the marine diatomSkeletonema costatum. A number of test technical factors as well as the variability in the sensitivity of natural phytoplankton have been investigated, using potassium dichromate as a reference toxicant in all tests. Some supplementary experiments were carried out with pentachlorophenol and with an industrial effluent. The sensitivity of the test increased with increasing incubation time, but for practical reasons 6 hours of incubation time is recommended.

Udgivelsesdato: 2005-Dec-15 We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.

Udgivelsesdato: 2006-May The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express beta-galactosidase (EC 3.2.1.23) at temperatures below 25 degrees C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a beta-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding beta-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of beta-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42 degrees C, but with short-term stability at temperatures above 25 degrees C.

Infectious bursal disease virus (IBDV) is a major cause of disease problems in the poultry industry and vaccination has therefore been applied intensively to control the infection. The classical methods of detection and characterization of IBDV are by the use of immunodiffusion test and histopathology. Since these methods are laborious and have low specificity alternatives are needed. In the present study, we report the development of a strain-specific multiplex RT-PCR technique, which can detect and differentiate between field strains of IBDV and vaccine virus strains including a so-called hot vaccine strain widely used in the European poultry industry. The method, which is highly specific, fast and inexpensive, can be applied in all laboratories with basal PCR capabilities and equipment.

The toxicity of two amines, nonylamine and decylamine, which have a narcotic effect on organisms, and ethylparathion (enzymatic inhibitor) on Daphnia magna has been studied when acting singly and in joint toxicity tests. This was done on the basis of an isobologram method where curves of constant response, i.e., isoboles are plotted vs. the concentrations of the two toxicants. The concentrations that immobilized 50, 10, and 0% of the test population in 48 h (IC50-48h, IC10-48h, and IC0-48h) were calculated. It is shown that nonylamine-decylamine mixtures follow a near concentration addition model of joint toxicity, while ethylparathion-decylamine mixtures follow a less than additive or near independent action model. The study was performed using three toxicity indices: additive index, sum of toxic units, and similarity parameter lambda. An analysis of the similarity parameter lambda is done to evaluate whether it has a constant value for IC50-48h, IC10-48h, and IC0-48h. The results suggest that it is quite probable that a constant value of lambda can be used to characterize all response levels, as the isoboles corresponding to the average value of the similarity parameter fit within the 95% confidence intervals of toxicant concentrations at all response levels. The average value of lambda is about 0.80 for the nonylamine-decylamine test and 0.30 for the ethylparathion-decylamine test.

Udgivelsesdato: 2003-Mar-25 Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli-a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.

The purpose of this study is to investigate the association of serum osteoprotegerin (OPG) and the A163G polymorphism in the OPG promoter with peripheral measures of bone mass and with odds ratios for wrist and hip fracture in a case-control study of postmenopausal Danish women. The study included 66 women with lower forearm fracture, 41 women with hip fracture, and 206 age-matched controls. All had broadband ultrasound attenuation (BUA) and speed of sound (SOS) measured at the heel as well as bone mineral density (BMD) measured by DXA at the distal forearm. S-OPG was measured by ELISA. The A163G genotypes were determined by PCR-RFLP analysis. S-OPG levels correlated positively with age ( r = 0.45; P << 0.0001) and negatively with distal forearm BMD ( r = -0.31; P << 0.0001), heel BUA ( r = -0.23; P << 0.0001), and heel SOS ( r = -0.22; P << 0.0001). Comparing the highest quartile of S-OPG to the lowest, the odds ratio for osteoporotic fracture was 2.5 (95% CI, 1.3-4.7; P = 0.006). The G allele of the A163G was associated with significantly lower t-scores of both lower forearm BMD, heel BUA, and heel SOS as well as being significantly more frequent in the fracture patients compared to the controls. Patients with a combination of the highest quartile of S-OPG and presence of the G allele ( n = 23) had a significantly elevated fracture odds ratio, 4.0 (95% CI, 1.7-9.9). A significant negative association between S-OPG with peripheral measures of bone mass and with increased fracture odds ratios was found. Furthermore, the A163G mutation in the OPG promoter had a significant influence on bone mass and fracture status independently of S-OPG level.

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.

Extrapolating toxicant effects with a fixed application factor (AF) approach or one of the species sensitivity distribution (SSD) models presumes that toxicant effects on single, individual-level endpoints reflect effects at the ecosystem level. Measured effect concentrations on plankton from multispecies field tests using tributyltin (TBT) and linear alkylbenzene sulfonates (LAS) were compared with published laboratory single-species test results and measured in situ concentrations. Extrapolation methods were evaluated by comparing predicted no-effect concentrations (PNECs), calculated by AF and SSD models with NOECs and E(L)C 50 s obtained from field studies. Overall, structural parameters were more sensitive than functional ones. Measured effect concentrations covered approximately the same range between laboratory and field experiments. Both SSD and AF approaches provide PNECs that appear to be protective for ecosystems. The AF approach is simpler to apply than the SSD models and results in PNECs that are no less conservative. Calculated PNEC values and the lowest field effect concentrations were lower than measured environmental concentrations for both substances, indicating that they may pose a risk to marine ecosystems.