Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions.

BACKGROUND: According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called 'minimum required performance limit' (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable. RESULTS: We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section 'Availability of supporting data' for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less. CONCLUSIONS: The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in -but not limited to- the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Does LDL-C determination method affect statin prescribing for primary prevention? A register-based study in Southern Denmark.

AIMS: Examine whether low-density lipoprotein cholesterol (LDL-C) determination method influences the rate of statin initiation for primary prevention of cardiovascular disease. METHODS AND RESULTS: We conducted a register-based retrospective study in the Region of Southern Denmark. Two hospital-based laboratories in the Region directly measure LDL-C whereas four laboratories calculate LDL-C using Friedewald's formula. Physicians do not choose which method is used. We included all statin-naïve patients ≥40 years with no history of cardiovascular disease, diabetes, or chronic kidney disease, who had their LDL-C determined during 2018-2019. There were 202,807 people who had LDL-C determined during the study period (median age 59 years, 44% women) of which 37% had a direct LDL-C measurement. The median reported LDL-C was 3.40 mmol/L (IQR 2.90 to 4.00) for those with a direct measurement versus 3.00 mmol/L (IQR 2.40 to 3.50) for those with calculated LDL-C. For those with direct measurement, re-calculated LDL-C (using Friedewald's formula) was 0.35 mmol/L lower than the reported direct LDL-C measurement. Among those with directly measured LDL-C, 3.6% initiated statins compared with 2.7% of those with a calculated LDL-C. Direct LDL-C measurement led to higher odds of having a statin initiated compared to calculated LDL-C (adjusted odds ratio 1.23, 95% CI 1.17 to 1.30); for those with triglycerides > 1.7 mmol/L the adjusted odds ratio was 1.41 (95% CI 1.30 to 1.52). CONCLUSION: Differences in the reporting of LDL-C from laboratories using different methods have a substantial influence on physician's decisions to prescribe statins.

Finite Element Simulation and Experimental Assessment of Laser Cutting Unidirectional CFRP at Cutting Angles of 45° and 90°.

Laser cutting of carbon fibre-reinforced plastics (CFRP) is a promising alternative to traditional manufacturing methods due to its non-contact nature and high automation potential. To establish the process for an industrial application, it is necessary to predict the temperature fields arising as a result of the laser energy input. Elevated temperatures during the cutting process can lead to damage in the composite's matrix material, resulting in local changes in the structural properties and reduced material strength. To address this, a three-dimensional finite element model is developed to predict the temporal and spatial temperature evolution during laser cutting. Experimental values are compared with simulated temperatures, and the cutting kerf geometry is examined. Experiments are conducted at 45° and 90° cutting angles relative to the main fibre orientation using a 1.1 mm thick epoxy-based laminate. The simulation accurately captures the overall temperature field expansion caused by multiple laser beam passes over the workpiece. The influence of fibre orientation is evident, with deviations in specific temperature data indicating differences between the estimated and real material properties. The model tends to overestimate the ablation rate in the kerf geometry, attributed to mesh resolution limitations. Within the parameters investigated, hardly any expansion of a heat affected zone (HAZ) is visible, which is confirmed by the simulation results.

First-time urea breath tests performed at home by 36,629 patients: a study of Helicobacter pylori prevalence in primary care.

BACKGROUND: The aim of the current study was (1) to describe the use of a (13) C-urea breath test (UBT) that was performed by patients at their homes as a part of a test-and-treat strategy in primary care and (2) to investigate the prevalence of Helicobacter pylori in patients taking a first-time UBT. MATERIAL AND METHODS: The patients performed UBTs at home based on the discretion of the general practitioner and mailed the breath bags to a central laboratory for analysis. Each patient was identified by a unique civil registration number. The study was population-based, and the background population was approximately 700,000 people. RESULTS: From 2003 to 2009, 44,487 UBTs were performed. Of these, 36,629 were first-time UBTs. In total, 726 of 45,213 breath bags received (1.6%) were unable to be analyzed because of errors with the bags. For both women and men who were ≤ 45 years of age, positive H. pylori declined over the time course of the study (women: 19.6% in 2003 to 17.6% in 2009, p < .01; men: 20.7% in 2003 to 16.9% in 2009, p < .001). Patients who were older than 45 years had significantly higher positive H. pylori results than younger patients. CONCLUSIONS: A test-and-treat system was possible to implement that allowed patients to perform UBTs at their homes. The results of the first-time UBTs demonstrated that approximately one of five patients who presented with dyspepsia in the clinical setting of Danish primary care was infected with H. pylori.

Interlayer Bonding Capability of Additively Manufactured Polymer Structures under High Strain Rate Tensile and Shear Loading.

Additive manufacturing of polymers via material extrusion and its future applications are gaining interest. Supporting the evolution from prototype to serial applications, additional testing conditions are needed. The additively manufactured and anisotropic polymers often show a weak point in the interlayer contact area in the manufacturing direction. Different process parameters, such as layer height, play a key role for generating the interlayer contact area. Since the manufacturing productivity depends on the layer height as well, a special focus is placed on this process parameter. A small layer height has the objective of achieving better material performance, whereas a larger layer height is characterized by better economy. Therefore, the capability- and economy-oriented variation was investigated for strain rates between 2.5 and 250 s-1 under tensile and shear load conditions. The test series with dynamic loadings were designed monitoring future applications. The interlayer tensile tests were performed with a special specimen geometry, which enables a correction of the force measurement. By using a small specimen geometry with a force measurement directly on the specimen, the influence of travelling stress waves, which occur due to the impact at high strain rates, is reduced. The interlayer tensile tests indicate a strain rate dependency of additively manufactured polymers. The capability-oriented variation achieves a higher ultimate tensile and shear strength compared to the economy-oriented variation. The external and internal quality assessment indicates an increasing primary surface profile and void volume content for increasing the layer height.

[Christmas article. Significantly fewer acute hospital admissions on days off].

The number of acute admissions in Danish hospitals is increasing and is causing recurrent overcrowding problems. There are fewer hospitalisations on days off, like weekends, public holidays and vacations, and this phenomenon is analysed in our review with data from the period 1.1.2008-31.12.2017. There were 20-30% fewer acute admissions on days off than on the about 60% normal working days in Denmark, and apparently without compensatory increases following the days off. Our findings suggest, that 1) doctors' indications for acute admissions cannot possibly be based on strict clinical criteria alone, and 2) the problem of growing hospitalisation and overcrowding could perhaps be attenuated by increasing the number of days off in the Danish healthcare system. However, further studies are required, to ensure that the quality of patient care is not adversely affected.

[Medication reconciliation].

The purpose of medicines reconciliation (MR) is to avoid medication errors through the complete and accurate transfer of information on patients' medicines during health care sector transitions. We review the rapidly expanding literature on MR showing a need for consensus on taxonomy and research into efficient ways to implement MR. Further, we describe quality improvement initiatives on MR in Denmark and challenge the, in our view, one-sided focus on information technology in MR.

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Diagnosis of Helicobacter Pylori Infection is Associated with Lower Prevalence and Subsequent Incidence of Crohn's Disease.

BACKGROUND AND AIMS: Helicobacter pylori infection may protect against some chronic inflammatory diseases. This study examined H. pylori infection and its association with the prevalence of the gastrointestinal diseases Crohn's disease [CD], ulcerative colitis [UC], and coeliac disease [CeD]. Incident cases in a follow-up period after H. pylori testing were recorded to investigate if protective effects of H. pylori persisted after probable eradication. METHODS: This was a historical cohort study performed in the Central Denmark Region. Patients were enrolled consecutively from primary health care centres after a urea breath test [UBT] for H. pylori and were then followed for a median of 6 years. The patient's diseases, country of birth, and gender were acquired from nationwide administrative registries. We used logistic regression to compare the prevalences of CD, UC, and CeD and Cox regression to compare the incidences of CD, UC, and CeD between H. pylori-positive and H. pylori-negative patients, adjusting for confounding variables. RESULTS: We found a lower prevalence of CD in H. pylori-positive than in H. pylori-negative patients (odds ratio = 0.36 [0.17-0.75]). There were fewer incident cases of CD in H. pylori-positive than H. pylori-negative patients in the follow-up period (hazard ratio = 0.59 [0.36-0.96]). Similar trends were found for CeD but not for UC. CONCLUSIONS: H. pylori infection may be a protective factor against the development of CD. However, the incidence of CD is still reduced after UBT and probable H. pylori eradication; thus, H. pylori status may be a marker for other factors that protect against CD.

The Arabidopsis thaliana chloroplast inner envelope protein ARTEMIS is a functional member of the Alb3/Oxa1/YidC family of proteins.

The Arabidopsis thaliana protein ARTEMIS is an integral component of the chloroplast inner envelope required for chloroplast division. It contains a domain of significant homology to members of the Alb3/Oxa1/YidC protein family. Here, we show that upon expression in yeast mitochondria, ARTEMIS can partially take over the function of yeast Oxa1 in the insertion and assembly of mitochondrial membrane proteins. This identifies ARTEMIS as a functional member of the Alb3/Oxa1/YidC protein family and suggests the existence of a novel protein sorting pathway in chloroplasts which integrates polypeptides from the stroma into the inner envelope by an evolutionary conserved process.

Text mining electronic health records to identify hospital adverse events.

Manual reviews of health records to identify possible adverse events are time consuming. We are developing a method based on natural language processing to quickly search electronic health records for common triggers and adverse events. Our results agree fairly well with those obtained using manual reviews, and we therefore believe that it is possible to develop automatic tools for monitoring aspects of patient safety.

[Patient deaths are not suited for monitoring quality and patient safety in hospitals]. / Dødsfald er ikke egnede til monitorering af kvalitet og patientsikkerhed i sygehusvæsenet.

Although some patient deaths in hospitals can be prevented if quality of care and patient safety are improved, it is not given that such deaths can be used to track improvements. Since preventable deaths only amount to about 5% of all deaths, a decline in that number will be a weak signal as far as mortality is concerned. The commonly used hospital standardised mortality ratio (HSMR) has a low signal-to-noise ratio and is therefore not suitable for monitoring. The same goes for structured reviews of health-care records of deceased patients. Consequently, a need to develop alternatives exists.

[Hospital standardised mortality ratios do not with certainty reflect the quality of patient care]. / Hospitalsstandardiseret mortalitetsratio kan ikke sikkert relateres til kvaliteten af patientbehandlingen.

Hospital standardised mortality ratios (HSMRs) are presumed to reflect an overall quality of patient care in hospitals. Figures for all Danish hospitals are being published quarterly on the Internet, and aggregated values are used to set goals for both regional and nationwide efforts to improve quality of care and patient safety. We have analysed the available data from 2007 through 2011 and cannot reconcile the quite substantial and often sudden changes in HSMRs with changes in quality of care, but believe that they are due to inherent noise in calculating HSMRs, e.g. owing to variable quality of the diagnostic coding.

[Uniform reference intervals and harmonised results in clinical biochemistry in Scandinavia--light ahead]. / Ens referenceintervaller og harmoniserede resultater inden for klinisk biokemi i de nordiske lande--der er lys forude.

Danish laboratories are introducing identical reference intervals for a number of biochemical components in accordance with a Nordic agreement and recommendations during 2007. Danish doctors will consequently experience both changes in reference interval limits as well as adjustments of patients' levels of reported results for some components.

Estimating the incidence of the acute coronary syndrome: data from a Danish cohort of 138 290 persons.

BACKGROUND: Estimates of incidence are crucial to the planning of public health measures, but most studies of incidence of, for example, acute myocardial infarction (MI) are troubled by methodological problems such as; (i) selection biases of the patients being included for study, (ii) lack of identification and control of the cohort under observation, (iii) inconsistencies in the use of diagnostic criteria, and (iv) missing data. We aimed to measure directly the incidence of the entire spectrum of the acute coronary syndrome (ACS), consisting of unstable angina pectoris, MI and sudden cardiac death (SCD), by use of the new criteria for MI as proposed in 2000. DESIGN: Cohort study. METHODS: From a cohort of 138 290 residents of the municipality of Aarhus, Denmark, aged 30-69 years, with a demographic structure known at the individual population member level, we prospectively identified all consecutive ACS patients from 1 April 2000 to 31 March 2002. The population was identified from Danish Population Registers. RESULTS: A total of 189 victims of SCD and 457 ACS patients who survived until admission to hospital were present. Consequently, crude incidence rate of ACS was 234 per 100 000 person-years. Unstable angina pectoris constituted for 16.9%, MI for 53.8% and SCD for 29.3% of ACS patients. CONCLUSIONS: Crude incidence rates of ACS were 137 and 331 per 100 000 person years for women and men, respectively. The incidence rate of ACS, as measured directly, was insignificantly 6% higher than expected from Danish administrative databases.

A second thylakoid membrane-localized Alb3/OxaI/YidC homologue is involved in proper chloroplast biogenesis in Arabidopsis thaliana.

The integral membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the thylakoid membrane of chloroplasts, the inner membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the thylakoid membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.

Expansins are involved in the formation of nematode-induced syncytia in roots of Arabidopsis thaliana.

Parasitism of the cyst nematode Heterodera schachtii is characterized by the formation of syncytial feeding structures in the host root. Syncytia are formed by the fusion of root cells, accompanied by local cell wall degradation, fusion of protoplasts and hypertrophy. Expansins are cell wall-loosening proteins involved in growth and cell wall disassembly. In this study, we analysed whether members of the expansin gene family are specifically and developmentally regulated during syncytium formation in the roots of Arabidopsis thaliana. We used PCR to screen a cDNA library of 5-7-day-old syncytia for expansin transcripts with primers differentiating between 26 alpha- and three beta-expansin cDNAs. AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA16, AtEXPA20 and AtEXPB3 could be amplified from the library. In a semi-quantitative RT-PCR and a Genechip analysis AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 were found to be upregulated specifically in syncytia, but not to be transcribed in surrounding root tissue. Histological analyses were performed with the aid of promoter::GUS lines and in situ RT-PCR. Results from both approaches supported the specific expression pattern. Among the specifically expressed genes, AtEXPA3 and AtEXPA16 turned out to be of special interest as they are shoot-specific in uninfected plants. We conclude that syncytium formation involves the specific regulation of expansin genes, indicating that the encoded expansins take part in cell growth and cell wall disassembly during syncytium formation.

The common polymorphism of apolipoprotein E: geographical aspects and new pathophysiological relations.

Apolipoprotein E (apoE) has important functions in systemic and local lipid transport, but also has other functions. The gene (APOE) shows a common polymorphism with three alleles--APOE*2, APOE*3, and APOE*4. Their frequencies vary substantially around the world, but APOE*3 is the most common almost everywhere and is often considered to be the ancestral or "wild-type" allele for that reason. However, there are several arguments for APOE*4 being the ancestral allele. But then, why has APOE*3 become so frequent? And why has APOE*4 not become extinct? The proportion of APOE*4 carriers increases from only 10-15% in southern Europe to 40-50% in the north. The gradient may be a trace of the demic expansion of agriculture that began about 10,000 years ago, but it may also reflect the possibility that APOE*4 carriers are less likely to develop vitamin D deficiency. The common APOE polymorphism is associated with varying risk of cardiovascular disease and Alzheimer's disease, but other interesting aspects may emerge in the future.

Twelve single nucleotide polymorphisms on chromosome 19q13.2-13.3: linkage disequilibria and associations with basal cell carcinoma in Danish psoriatic patients.

The genetic susceptibility to basal cell carcinoma (BCC) among Danish psoriatic patients was investigated in association studies with 12 single nucleotide polymorphisms on chromosome 19q13.2-3. The results show a significant association between BCC and the A-allele of a polymorphism in ERCCI exon4 (Odds ratio 12;95% Confidence Interval 1.17-124; p(chi2, two-side) = 0.019) and to a lesser extent with XPD exon6 (p = 0.06). This is in accordance with recent studies of a different group of BCC cases (Rockenbauer et al. (in press) Carcinogenesis; Yin et al. (manuscript submitted for publication). Cancer Epidemiol. Biomarkers Prev), which places two highly influential markers between these two genes. The analysis also confirmed that considerable linkage disequilibrium exists between SNPs both within genes and between genes in this region. The combined studies suggest that genetic variation in nucleotide excision repair is of importance for the development of BCC.