Are the latest point-of-care D-dimer devices ready for use in general practice? A prospective clinical evaluation of five test systems with a capillary blood feature for suspected venous thromboembolism.

INTRODUCTION: We evaluated clinical performance of five novel point-of-care (POC) D-dimer devices with a capillary finger stick feature for predicting venous thromboembolism (VTE) in general practice: Exdia TRF Plus (E), AFIAS-1® (A), Standard F200® (S), LumiraDx™ (L) and Hipro AFS/1® (H). MATERIALS AND METHODS: Primary care patients with a low suspicion of a VTE were asked to consent to (i) draw additional venous blood samples, (ii) perform a capillary POC D-dimer test, (iii) approach their general practitioner afterwards for clinical outcomes. Venous plasma samples were processed on all POC devices and a laboratory-based assay (STA-Liatest®D-Di PLUS assay). Results were compared with clinical outcomes to generate performance characteristics. Capillary and venous blood results were used for a matrix comparison. RESULTS: Venous plasma samples from 511 participants, of whom 57 had VTE, were used for clinical performance analyses. Areas under Receiving Operating Characteristic Curves ranged from 0.90 (95 % CI: 0.86-0.94) (H) to 0.93 (0.90-0.96) (E). All false-negative rates were below 1.4 % (95 % CI: 0.5 %-3.4 %). Matrix comparison demonstrated correlation coefficients ranging from r = 0.11 (95 % CI: -0.15-0.36) (H) to r = 0.94 (0.90-0.97) (A) with concordance percentages ranging from 71.4 % (applying a D-dimer cutoff of 500 ng/mL) (H) to 100 % (applying an age-dependent D-dimer cutoff) (A). CONCLUSIONS: Clinical performance of the POC D-dimer devices for predicting a VTE in low-risk patients was comparable to that of a laboratory-based assay. However, our results indicate that the finger stick feature of certain devices should be further improved. (NL71809.028.19.).

Two point-of-care test-based approaches for the exclusion of deep vein thrombosis in general practice: a cost-effectiveness analysis.

BACKGROUND: In the diagnostic work-up of deep vein thrombosis (DVT), the use of point-of-care-test (POCT) D-dimer assays is emerging as a promising patient-friendly alternative to regular D-dimer assays, but their cost-effectiveness is unknown. We compared the cost-effectiveness of two POCT-based approaches to the most common, laboratory-based, situation. METHODS: A patient-level simulation model was developed to simulate the diagnostic trajectory of patients presenting with symptoms of DVT at the general practitioner (GP). Three strategies were defined for further diagnostic work-up: one based on current guidelines ('regular strategy') and two alternative approaches where a POCT for D-dimer is implemented at the 1) phlebotomy service ('DVT care pathway') and 2) GP practice ('fast-POCT strategy'). Probabilities, costs and health outcomes were obtained from the literature. Costs and effects were determined from a societal perspective over a time horizon of 6 months. Uncertainty in model outcomes was assessed with a one-way sensitivity analysis. RESULTS: The Quality-Adjusted Life Years (QALYs) scores for the three DVT diagnostic work-up strategies were all around 0.43 across a 6 month-time horizon. Cost-savings of the two POCT-based strategies compared to the regular strategy were €103/patient for the DVT care pathway (95% CI: -€117-89), and €87/patient for the fast-POCT strategy (95% CI: -€113-67). CONCLUSIONS: Point-of-care-based approaches result in similar health outcomes compared with regular strategy. Given their expected cost-savings and patient-friendly nature, we recommend implementing a D-dimer POCT device in the diagnostic DVT work-up.

Geometric localization in supported elastic struts.

Localized deformation patterns are a common motif in morphogenesis and are increasingly finding applications in materials science and engineering, in such instances as mechanical memories. Here, we describe the emergence of spatially localized deformations in a minimal mechanical system by exploring the impact of growth and shear on the conformation of a semi-flexible filament connected to a pliable shearable substrate. We combine numerical simulations of a discrete rod model with theoretical analysis of the differential equations recovered in the continuum limit to quantify (in the form of scaling laws) how geometry, mechanics and growth act together to give rise to such localized structures in this system. We find that spatially localized deformations along the filament emerge for intermediate shear modulus and increasing growth. Finally, we use experiments on a 3D-printed multi-material model system to demonstrate that external control of the amount of shear and growth may be used to regulate the spatial extent of the localized strain texture.

High pneumococcal DNA load, procalcitonin and suPAR levels correlate to severe disease development in patients with pneumococcal pneumonia.

Community-acquired pneumonia (CAP) is mostly caused by Streptococcus pneumoniae. Identification of the pathogen causing CAP can be achieved by conventional culture techniques of sputum and/or blood, antigen detection from urine or molecular analysis. However, it remains difficult to determine patients who are at risk of severe disease development (intensive care unit [ICU] admittance and/or death). In this retrospective study, 121 patients admitted to the emergency department with pneumonia symptoms were included. Several markers of infection (pneumococcal DNA load in blood (real-time LytA PCR), white blood cell (WBC) count, C-reactive protein (CRP), procalcitonin (PCT) and soluble urokinase plasminogen activator receptor (suPAR) levels) were assessed for their ability to predict severe disease development. Of 121 patients, 6 were excluded from the study because of an alternative diagnosis, whereas 8 were excluded from biomarker analysis because of the presence of co-morbidities. Of the 115 patients analysed by the LytA PCR, 23 were positive. PCR detected S. pneumoniae DNA in 82% of patients with positive blood culture for S. pneumoniae. PCR missed three samples from patients in which S. pneumoniae was recovered by blood cultures. However, eight additional LytA PCR-positive samples were detected from patients whose blood cultures remained negative. Pneumococcal DNA load was also monitored in time for 31 patients, of whom 11 had positive PCR results. For 10 out of 11 (91%) positive PCR patients, a clear increase in Ct-values was observed, indicating a lower pneumococcal DNA load in the blood as a result of antibiotic therapy. Biomarker analysis was performed in 107 patients, of whom 29 showed severe disease development. Pneumococcal DNA load (p = 0.026), PCT (p = 0.046) and suPAR (p = 0.001) levels most reliably predicted severe disease development. In conclusion, in patients with CAP, higher pneumococcal DNA load, PCT and suPAR values are associated with severe disease development (ICU admission and/or death). These biomarkers may be useful tools for triage of patients suspected of having CAP in the emergency department.

Biophysical model of the role of actin remodeling on dendritic spine morphology.

Dendritic spines are small membranous structures that protrude from the neuronal dendrite. Each spine contains a synaptic contact site that may connect its parent dendrite to the axons of neighboring neurons. Dendritic spines are markedly distinct in shape and size, and certain types of stimulation prompt spines to evolve, in fairly predictable fashion, from thin nascent morphologies to the mushroom-like shapes associated with mature spines. It is well established that the remodeling of spines is strongly dependent upon the actin cytoskeleton inside the spine. A general framework that details the precise role of actin in directing the transitions between the various spine shapes is lacking. We address this issue, and present a quantitative, model-based scenario for spine plasticity validated using realistic and physiologically relevant parameters. Our model points to a crucial role for the actin cytoskeleton. In the early stages of spine formation, the interplay between the elastic properties of the spine membrane and the protrusive forces generated in the actin cytoskeleton propels the incipient spine. In the maturation stage, actin remodeling in the form of the combined dynamics of branched and bundled actin is required to form mature, mushroom-like spines. Importantly, our model shows that constricting the spine-neck aids in the stabilization of mature spines, thus pointing to a role in stabilization and maintenance for additional factors such as ring-like F-actin structures. Taken together, our model provides unique insights into the fundamental role of actin remodeling and polymerization forces during spine formation and maturation.

Blood warming, pump heating and haemolysis in low-flow extracorporeal life support; an in vitro study using freshly donated human blood.

Low-flow extracorporeal life support can be used for cardiopulmonary support of paediatric and neonatal patients and is also emerging as a therapy for patients suffering from exacerbation of chronic obstructive pulmonary disease. However, pump heating and haemolysis have proven to negatively affect the system and outcome. This in vitro study aimed at gaining insight into blood warming, pump heating and haemolysis related to the performance of a new low-flow centrifugal pump. Pump performance in the 400-1,500 ml/min flow range was modulated using small-sized dual-lumen catheters and freshly donated human blood. Measurements included plasma free haemoglobin, blood temperature, pump speed, pump pressure, blood flow and thermographic imaging. Blood warming (ΔTmax=0.5°C) had no relationship with pump performance or haemolysis (R2max=0.05). Pump performance-related parameters revealed no relevant relationships with haemolysis (R2max=0.36). Thermography showed no relevant heat zones in the pump (Tmax=36°C). Concerning blood warming, pump heating and haemolysis, we deem the centrifugal pump applicable for low-flow extracorporeal circulation.

Spatio-temporal dynamics of expansion growth in roots: automatic quantification of diurnal course and temperature response by digital image sequence processing.

A newly developed technique based on image sequence analysis allows automatic and precise quantification of the dynamics of the growth velocity of the root tip, the distribution of expansion growth rates along the entire growth zone and the oscillation frequencies of the root tip during growth without the need of artificial landmarks. These three major parameters characterizing expansion growth of primary roots can be analysed over several days with high spatial (20 microm) and temporal resolution (several minutes) as the camera follows the growing root by an image-controlled root tracking device. In combination with a rhizotron set up for hydroponic plant cultivation the impact of rapid changes of environmental factors can be assessed. First applications of this new system proved the absence of diurnal variation of root growth in Zea mays under constant temperature conditions. The distribution profile of relative elemental growth rate (REGR) showed two maxima under constant and varying growth conditions. Lateral oscillatory movements of growing root tips were present even under constant environmental conditions. Dynamic changes in velocity- and REGR-distribution within 1 h could be quantified after a step change in temperature from 21 degrees C to 26 degrees C. Most prominent growth responses were found in the zone of maximal root elongation.

Towards decision support for waiting lists: an operations management view.

This paper considers the phenomenon of waiting lists in a healthcare setting, which is characterised by limitations on the national expenditure, to explore the potentials of an operations management perspective. A reference framework for waiting list management is described, distinguishing different levels of planning in healthcare--national, regional, hospital and process--that each contributes to the existence of waiting lists through managerial decision making. In addition, different underlying mechanisms in demand and supply are distinguished, which together explain the development of waiting lists. It is our contention that within this framework a series of situation specific models should be designed to support communication and decision making. This is illustrated by the modelling of the demand for cataract treatment in a regional setting in the south-eastern part of the Netherlands. An input-output model was developed to support decisions regarding waiting lists. The model projects the demand for treatment at a regional level and makes it possible to evaluate waiting list impacts for different scenarios to meet this demand.

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross-linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.

The functioning of the SRP receptor FtsY in protein-targeting in E. coli is correlated with its ability to bind and hydrolyse GTP.

In this study, we have established that FtsY, the E. coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY. These observations suggest that in E. coli GTP is important for proper functioning of FtsY in protein-targeting.

A genetic diagnostic survey in an institutionalized population of 116 moderately to severely retarded male patients: the Rekem experience.

In this paper we report the results of a genetic-diagnostic survey of 116 institutionalized male patients who were moderately to severely retarded. In 31 patients (26.7%) a constitutional cause of their mental impairment was found: chromosomal abnormalities in 11 patients (9.5%), Mendelian disorders in 16 (13.8%), of which 8 fragile X patients (6.9%), and a MCA/MR syndrome in 4 patients (3.4%). Acquired forms of mental retardation occurred in 26 patients (22.4%): CNS-dysfunction due to pre-, peri- or postnatal causes were most likely in 18 patients (15.5%), while infections played a major role in 8 (6.9%). In 59 patients (50.9%) not etiological diagnosis could be made.

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY.

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha-subunit of the eukaryotic SRP receptor ('docking protein') in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta-lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY-depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre-beta-lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.

A dual role for phosphatidylglycerol in protein translocation across the Escherichia coli inner membrane.

The involvement of phosphatidylglycerol in the SecA-independent translocation of M13 procoat in Escherichia coli was demonstrated. Processing of procoat to mature coat protein was retarded when the level of phosphatidylglycerol was reduced. In vitro translocation experiments using inner membrane vesicles isolated from a strain with inducible synthesis of phosphatidylglycerol, showed that translocation of procoat and of a SecA-dependent procoat analog was proportional to the content of phosphatidylglycerol. Moreover, introduction of phosphatidylglycerol by means of a lipid transfer method into phosphatidylglycerol-depleted inner membrane vesicles, efficiently restored procoat translocation. The phosphatidylglycerol dependence in both the SecA-dependent and -independent translocation pathway indicates that phosphatidylglycerol plays a dual role in translocation. We suggest that besides membrane binding of SecA this lipid has a direct interaction with the M13 procoat in translocation across the inner membrane.

OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane.

Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a hydrophobic core of varying length. It is shown that there is a direct relationship between the phosphatidylglycerol requirement of translocation and the requirement within the translocation process for positive charges on the signal sequence. This provides further evidence that the negative head group of the lipid is required for functional interaction with the positively charged N terminus of the signal sequence.

In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex.

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.

Elevated cytosolic concentrations of SecA compensate for a protein translocation defect in Escherichia coli cells with reduced levels of negatively charged phospholipids.

Cellular extracts from cells with reduced synthesis of negatively charged phospholipids were found to support in vitro translocation of the precursor of the outer membrane protein PhoE with increased efficiency. Analysis of these extracts revealed that they contain increased levels of SecA. SecA depletion resulted in a loss of the translocation stimulatory activity, which could be restored by re-addition of purified SecA. We conclude that elevated cytosolic levels of SecA counteract the reduction of translocation efficiency due to low levels of negatively charged phospholipids in the inner membrane.

Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes.

Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R. L., Dowhan, W., Tommassen, J., and De Kruijff, B. (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280). Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles. Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase.