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AbstractIn oncology 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG), a glucose analogue, is the most used positron emission tomography (PET) tracer. There are however some limitations due to low metabolic activity or high surrounding physiological uptake in several tumors or regions. Investigating new tracers or methods is expensive and elaborative when animal experiments or phase I clinical trials are used. In vitroexperiments can overcome these limitations. We analyzed the influence of incubation time, cell medium conditions, administered activity, and cell density on [18F]-FDG uptake in six different cell cultures. Glucose transporter 1 (GLUT1)- and hexokinase 2 (HK2)-expression at high and low cell density was analyzed using immunocytochemistry. FDG-uptake increases over time and absence of glucose in the incubation medium increases uptake. By increasing the administered activity, uptake per protein also increases and tracer uptake per protein is lower at higher cell densities. Immunocytochemical analysis reveals a lower expression of both GLUT1 and HK2 at higher cell concentrations. All investigated parameters influenced FDG uptake and therefore we can conclude it is of utmost importance to keep administered activity, incubation medium, and time constant and to correct uptake when cell density changes due to environmental conditions, such as therapy. [ABSTRACT FROM AUTHOR]

Purpose To give an up-to-date overview of the potential clinical utility of 18F-labelled choline derivatives for tumour imaging with positron emission tomography. Methods A PubMed search for 18F-labelled choline analogues was performed. Review articles and reference lists were used to supplement the search findings. Results 18F-labelled choline analogues have been investigated as oncological PET probes for many types of cancer on the basis of enhanced cell proliferation. To date, studies have focused on the evaluation of prostate cancer. Available studies have provided preliminary results for detecting local and metastatic disease. Experience with 18F-fluorocholine PET in other tumour types, including brain and liver tumours, is still limited. In the brain, excellent discrimination between tumour and normal tissue can be achieved due to the low physiological uptake of 18F-fluorocholine. In the liver, in which there is a moderate to high degree of physiological uptake in normal tissue, malignancy discrimination may be more challenging. Conclusion PET/CT with 18F-fluorocholine can be used to detect (recurrent) local prostate cancer, but seems to have limited value for T (tumour) and N (nodal) staging. In patients presenting with recurrent biochemical prostate cancer, it is a suitable single-step examination with the ability to exclude distant metastases when local salvage treatment is intended. In the brain, high-grade gliomas, metastases and benign lesions can be distinguished on the basis of 18F-fluorocholine uptake. Moreover, PET imaging is able to differentiate between radiation-induced injury and tumour recurrence. In the liver, 18F-fluorocholine PET/CT seems promising for the detection of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Background: The increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC. Methods: Weekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC. Results: The N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets. [ABSTRACT FROM AUTHOR]

The discovery that antiphospholipid antibodies recognize plasma proteins that bind to phospholipids rather than recognizing phospholipids themselves has been a major advance in research into antiphospholipid syndrome (APS). It is now established that β2-glycoprotein I (β2GPI) is the most important antigen for antiphospholipid antibodies. However, the possible pathologic mechanism is still much debated. This is mainly because not all patients with anti-β2GPI antibodies show clinical symptoms that are related to APS. Several reports indicate that anti-β2GPI antibodies with lupus anticoagulant (LA) activity are clinically of much importance. Most patients with LA caused by anti-β2GPI antibodies suffer from thrombosis as a result of recognition of the first domain of β2GPI by these antibodies. In the search for a pathologic mechanism that might explain the high occurrence of thrombosis in patients with anti-domain I antibodies (LA-causing anti-β2GPI antibodies), it was found that these antibodies show increased resistance to the anticoagulant activity of annexin A5. We have shown that the same population of antibodies also displays increased resistance to activated protein C. Owing to the diversity of clinical symptoms related to APS, it is likely that other pathologic mechanisms also contribute to the occurrence of APS-related symptoms. [ABSTRACT FROM AUTHOR]

Sub-pixel mapping and sub-pixel sharpening are techniques for increasing the spatial resolution of sub-pixel image classifications. The proposed method makes use of wavelets and artificial neural networks. Wavelet multiresolution analysis facilitates the link between different resolution levels. In this work a higher resolution image is constructed after estimation of the detail wavelet coefficients with neural networks. Detail wavelet coefficients are used to synthesize the high-resolution approximation. The applied technique allows for both sub-pixel sharpening and sub-pixel mapping. An algorithm was developed on artificial imagery and tested on artificial as well as real synthetic imagery. The proposed method resulted in images with higher spatial resolution showing more spatial detail than the source imagery. Evaluation of the algorithm was performed both visually and quantitatively using established classification accuracy indices. [Copyright &y& Elsevier]

Develops a state-space behavioral model for complementary metal oxide semiconductors class E power amplifiers. Design of amplifiers; Use of state-space equations; Derivation of the power balance of the power amplifier; Design of switched power amplifiers.

Investigates the functional role of vitamin K-dependent protein human factor IX insertion loop 256-268, and in particular of residues Asn264 and Lys265 therein. Effect of mutation of residues Asn264-Lys265 on the interactions in the S2-binding site; Ability of loop 256-268 to restrict the enzymatic activity of factor IXa.

The article discusses a research study on the membrane interactive domains of blood clotting factors VIII and V. It mentions that the 3- dimensional structures of these domains are referred as fatty feet which mediate the interaction with lipid membranes. Swapping the domains between factor VIII and factor V resulted in remarkable change in lipid binding and biological activity.

• A proximal sensing platform was built and evaluated for in-field hyperspectral imaging. • 750 nm, 550 nm and 680 nm were most discriminating for Alternaria solani detection. • Pixel based accuracy was 0.92, object based precision was 0.22 and recall 0.84. Automatic detection of early blight caused by Alternaria solani could promote a drastic reduction in the consumption of plant protection agents and the related production losses. A proximal sensing platform was constructed and calibrated for acquiring high resolution hyperspectral images in the field, and used to accurately map Alternaria lesions. High resolution canopy reflectance images were obtained for 32 potato plants that had been infected with A. solani and 32 healthy reference plants. Spectral classifiers like partial least squares discriminant analysis (PLS-DA) and support vector machines (SVM) based on PCA scores were tested to discriminate affected and non-affected pixels. Both spectral classifiers performed well at pixel level with accuracies above 0.92. The NIR region (750 nm) was identified as the most discriminative part of the spectrum for detecting the lesions. As the disease pressure is typically expressed as the number of lesions per area, the accuracy was also evaluated at this level. This indicated a considerable number of false detections at the edges of the leaves and the leaf axils. Therefore, a decision tree was designed based on expert knowledge about the shape of Alternaria lesions, and used to post-process the classified images. This reduced the number of false detections, increasing the precision from 0.17 to 0.22 at the expense of a reduction in recall from 0.88 to 0.84. This leaves considerable room for improvement in the classification accuracy at the object level. We learned that (1) few, broad wavelengths are sufficient and (2) spatial context is essential for the detection of lesions caused by Alternaria infection. The application of more powerful object classification techniques such as convolutional neural networks to enhance the model performance by efficiently encapsulating the spatial context in the classifier might further improve the detection performance. This could pave the way to UAV or tractor based Alternaria mapping. [ABSTRACT FROM AUTHOR]

HABP2 (hyaluronan-binding protein 2) is a Ca2+-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affectsHABP2function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+- dependent, with Km decreasing from 3.0 to 0.6 mM upon titration from 0 to 0.3 M Na+. In the presence of 5 mM Ca2+, Km was further reduced to 0.05 mM, but without an appreciable contribution of Na+. At physiological concentrations of Na+ and Ca2+, the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity. [ABSTRACT FROM AUTHOR]

Precise application of granular fertiliser requires the correct calibration of spreading equipment. Currently, the performance of spreaders is assessed by determining the transverse distribution using a row of collection trays aligned perpendicularly to the driving direction. When the homogeneity of the distribution for a given spreader with a given fertilizer is inadequate, the spreader settings need to be corrected. However, because particle deposition is measured in only one dimension, this technique does not provide an adequate insight into the spreading process as a whole. The distribution should be measured in two dimensions, which, due to the large spreading widths involved with modern spreaders, is only possible by sampling the spreading area. In this paper, two different two-dimensional methods to determine spreading pattern were evaluated, each consisting of a sampling method and a matching interpolation algorithm. Both sampling methods use a similar number of collecting trays. Experiments were executed using three commonly used types of fertiliser (CAN (Ammonium-nitrate fertiliser), NPK (Compound fertiliser), KCl (Potassium-chloride fertiliser)) with different physical properties. The resulting spreading patterns were compared with the standard one-dimensional technique. Differences were found both in the application rate and application homogeneity. The results illustrated the importance of using an adequate spreading pattern measurement technique to accurately compare the spreading patterns of granular fertiliser spreaders. [ABSTRACT FROM AUTHOR]

Better characterization of the fertilizer spreading process, especially the fertilizer pattern distribution on the ground, requires an accurate measurement of individual particle properties and dynamics. Both 2D and 3D high speed imaging techniques have been developed for this purpose. To maximize the accuracy of the predictions, a specific illumination level is required. This paper describes the development of a high irradiance LED system for high speed motion estimation of fertilizer particles. A spectral sensitivity factor was used to select the optimal LED in relation to the used camera from a range of commercially available high power LEDs. A multiple objective genetic algorithm was used to find the optimal configuration of LEDs resulting in the most homogeneous irradiance in the target area. Simulations were carried out for different lenses and number of LEDs. The chosen configuration resulted in an average irradiance level of 452 W/m2 with coefficient of variation less than 2%. The algorithm proved superior and more flexible to other approaches reported in the literature and can be used for various other applications. [ABSTRACT FROM AUTHOR]

ABSTRACT BACKGROUND Multimodality imaging such as proton magnetic resonance spectroscopy (MRS) and positron emission tomography (PET) have provided information specific to the underlying mechanisms of many brain diseases, including multiple sclerosis (MS). PURPOSE To determine the structural and metabolic characterization of two particular variants of MS, namely tumefactive MS and Balo's concentric sclerosis (BCS). METHODS Conventional MR imaging, diffusion and perfusion MR, MR spectroscopy and PET imaging with F-18 fluorodeoxyglucose (FDG) and F-18 fluoromethylcholine (FCho) were performed. RESULTS In a case with pathologically proven tumefactive MS, magnetic resonance imaging (MRI) showed a pseudotumoral lesion with incomplete ring enhancement, peripheral diffusion restriction, and high choline and lactate peaks on MRS. On follow-up, the lesion showed significant growth. In a case of BCS, MRI showed an onion-like lesion without contrast enhancement or diffusion restriction, and only a moderate increase in choline on MRS. The lesion remained stable on follow-up. On PET, there was no uptake of F-18 FDG in either type of MS lesion. Conversely, uptake of F-18 FCho was moderate in tumefactive MS, whereas no F-18 FCho uptake was noted in the lesion with, on MRI, typical features of BCS. CONCLUSIONS Our findings illustrate that metabolic features may differ between variants of MS possibly signifying different disease activity. [ABSTRACT FROM AUTHOR]

In perennial ryegrass breeding programmes, dry‐matter yield (DMY) of individual plots is monitored destructively at the different cuts or derived from non‐destructive canopy height measurements using devices like rising plate meters (RPM). These approaches both have constraints. Destructive sampling implies low temporal resolution, restraining the study of dry‐matter accumulation rates, while RPM measurements are influenced by the canopy structure and limit intra‐field variability identification. We present a phenotyping methodology, based on the use of an affordable RGB camera mounted on an unmanned aerial vehicle (UAV), to monitor the spatial and temporal evolution of canopy height and to estimate DMY. Weekly flights were carried out from April to October above a field comprising a diverse set of accessions. To test the capacity of UAV imagery to estimate canopy height, 8 ground control points and 28 artificial height references were placed at different locations. Accurate flights with an RMSE as low as 0.94 cm were achieved. In addition, canopy height was recorded using an RPM and destructive biomass samples were collected. Different models (linear, multiple linear, principal components, partial least squares regression and random forest) were used to predict DMY, and their performance was evaluated. The best estimations were obtained by combining variables including canopy height, vegetation indices and environmental data in a multiple linear regression (R2 = .81). All models built using UAV data obtained a lower RMSE than the one using RPM data. The approach presented is a possibility for breeders to incorporate new information in their selection process. [ABSTRACT FROM AUTHOR]

Abstract The vascular endothelium provides a unique interaction plane for plasma proteins and leukocytes in inflammation. The pro-inflammatory cytokines Tumor Necrosis Factor α (TNFα) and interleukin 1β (IL-1β) have a profound effect on endothelial cells, which includes increased levels of adhesion molecules and a disrupted barrier function. To assess the endothelial response to these cytokines at the protein level, we evaluated changes in the whole proteome, cell surface proteome and phosphoproteome after 24 h of cytokine treatment. The effects of TNFα and IL-1β on endothelial cells were strikingly similar and included changes in proteins not previously associated with endothelial inflammation. Temporal profiling revealed time-dependent proteomic changes, including a limited number of early responsive proteins such as adhesion receptors ICAM1 and SELE. In addition, this approach uncovered a greater number of late responsive proteins, including proteins related to self-antigen peptide presentation, and a transient increase in ferritin. Peptide-based cell surface proteomics revealed extensive changes at the cell surface, which were in agreement with the whole proteome. In addition, site-specific changes within ITGA5 and ICAM1 were detected. Combined, our integrated proteomic data provide detailed information on endothelial inflammation, emphasize the role of the extracellular matrix therein, and include potential targets for therapeutic intervention. Significance Pro-inflammatory cytokines induce the expression of cell adhesion molecules in vascular endothelial cells. These molecules mediate the adhesion and migration of immune cells across the vessel wall, which is a key process to resolve infections in the underlying tissue. Dysregulation of endothelial inflammation can contribute to vascular diseases and the vascular endothelium is therefore an attractive target to control inflammation. Current strategies targeting endothelial adhesion molecules, including PECAM, CD99, ICAM1 and VCAM1 do not completely prevent transmigration. To identify additional therapeutic targets, we mapped the endothelial proteome after pro-inflammatory cytokine treatment. In addition to the whole proteome, we assessed the surface proteome to focus on cell adhesion molecules, and the phosphoproteome to uncover protein activation states. Here, we present an integrated overview of affected processes which further improves our understanding of endothelial inflammation and may eventually aid in therapeutic intervention of imbalanced inflammation. Graphical abstract Unlabelled Image Highlights • TNFα and IL-1β induce strikingly similar proteomic profiles in vascular endothelium • Distinction between early and late onset of proteomic changes • Extensive cell surface changes including potential therapeutic targets • Integrated analysis of biological processes, including the extracellular matrix • Cell surface proteomics detects intramolecular changes in ITGA5 and ICAM1 [ABSTRACT FROM AUTHOR]

The D0-D3 fragment of von Willebrand factor (VWF) can be divided into TIL0-E0-VWD3-C8_3-TIL3-E3 subdomains of which TIL0-E0-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D0, D3 and monomeric C-terminal subdomain truncation variants of D0-D3. Competitive binding assays and surface plasmon resonance analysis revealed that D0 requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D0-D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D0-D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D0-D3 is indispensable for effective interaction between D0 and FVIII explaining why specific mutations in D3 can impair FVIII binding. [ABSTRACT FROM AUTHOR]

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2 M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2 M using U87MG cells. The results show that α2 M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091–2095 and 2155–2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding. [ABSTRACT FROM AUTHOR]

Centrifugal fertilizer spreaders are by far the most commonly used granular fertilizer spreader type in Europe. Their spread pattern however is error-prone, potentially leading to an undesired distribution of particles in the field and losses out of the field, which is often caused by poor calibration of the spreader for the specific fertilizer used. Due to the large environmental impact of fertilizer use, it is important to optimize the spreading process and minimize these errors. Spreader calibrations can be performed by using collection trays to determine the (field) spread pattern, but this is very time-consuming and expensive for the farmer and hence not common practice. Therefore, we developed an innovative multi-camera system to predict the spread pattern in a fast and accurate way, independent of the spreader configuration. Using high-speed stereovision, ejection parameters of particles leaving the spreader vanes were determined relative to a coordinate system associated with the spreader. The landing positions and subsequent spread patterns were determined using a ballistic model incorporating the effect of tractor motion and wind. Experiments were conducted with a commercial spreader and showed a high repeatability. The results were transformed to one spatial dimension to enable comparison with transverse spread patterns determined in the field and showed similar results. [ABSTRACT FROM AUTHOR]

As lameness detection in dairy cattle using visual locomotion scoring is cumbersome and subjective, research efforts are dedicated to develop automatic lameness detection systems. ‘Tender hoof placement’ and the distribution of the body weight over the four legs are possible lameness indicators, but no research exists on how to derive from automatically measured gait characteristics. This study aims to derive new variables related to the (i) landing, full weight bearing and lifting phases of a stance time and the (ii) time spent on combinations of legs during the different phases of the gait cycle from cow gait recordings on a dedicated pressure mat, known as the Gaitwise. Data of 9 non-lame, 11 mildly lame and 12 severely lame cows were gathered. For all measurements, each variable was calculated per leg or combination of legs, after which the group means of each variable were compared between the three lameness statuses using a one-way ANOVA analysis. Landing and lifting variables indicated that the proportion of time for hoof placement and hoof lifting during the total stance time was longer in lame cows, and that the proportion of full weight bearing time was shorter. Lame cows were thus more careful to place and retract the hind feet in the case of a hind-lame leg. Support time variables indicated that lame cows increased the percentage triple support time (i.e time spent with three feet on the ground during walking) and lowered the percentage double support (i.e. time spent with two legs on the ground). Also, double support combinations on the same side of the body were preferred above diagonal combinations. The newly defined gait variables indeed reflect tenderness of hoof placement and body weight distribution and hence seem useful for discriminating between non-lame, mildly lame and severely lame cows. However, several of these interesting variables may have to be combined to obtain automatic lameness detection with sufficient accuracy. [ABSTRACT FROM AUTHOR]

While previous research has shown the potential of automatic lameness detection by means of a pressure mat, these systems are currently not adopted in practice due to their high cost and low on-farm applicability. Therefore, the aim of this study was to investigate to what level the size (0.61 × 4.88 m) and resolution (0.0127 × 0.0127 m) of the pressure mat can be reduced without significant loss in lameness detection performance. To this end, standard gait variables were calculated based on adapted datasets in which the available data had been reduced to simulate the effects of a decreasing mat length and sensor resolution. These extracted gait variables were then used in a linear discriminant analysis to classify cows as non-lame, mildly lame or severely lame. This analysis indicated that the measurement zone length must be at least 3.28 m to successfully monitor one complete gait cycle, while the size of each individual sensing element should not be larger than 2.58 ∗ 10 −3 m 2 to avoid an increase in the misidentification of imprints. When these limits were taken into account, the obtained overall lameness detection accuracy was not worse than that of the original system. [ABSTRACT FROM AUTHOR]

LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteinerich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system. [ABSTRACT FROM AUTHOR]

With the increase in working widths for applicators, granular fertiliser particles spread by centrifugal spreaders have more extensive airborne trajectories. In the field, particles can be subjected to wind which can cause their trajectories to change. In this paper, a 3D ballistic model was developed describing the motion of fertiliser particles taking wind effects into account. The physical properties of eight commonly used fertiliser types were determined: particle density, size distribution and angle of repose. X-ray micro computed tomography was used to determine the shape of the particles and estimate the corresponding drag coefficient. Using these parameters in the newly derived ballistic model, the effect of wind on individual fertiliser trajectories was quantified for each fertiliser type. Three wind directions (head- tail- and crosswind) were analysed for two windspeeds: 3 and 6 m s −1 . The magnitude of the effect was strongly dependent on the physical properties of the fertiliser type, the windspeed and wind direction. Simulations showed that a windspeed of 3 m s −1 already can affect the landing position of individual fertiliser particles by several metres. Generally, particles with lower density, smaller size and a more irregular shape were more sensitive to wind. [ABSTRACT FROM AUTHOR]

Changes in the feeding pattern of a pig may indicate disease or welfare issues and can also be strongly related to productivity changes. Automatic measurements of the feeding patterns of group-housed growing-finishing pigs can enable the detection of such feeding related changes. In this paper, a high frequency (HF) Radio Frequency Identification (RFID) system was used to register pigs’ feeding patterns at a commercial-type feed trough. The raw RFID registrations had to be translated into feeding visits, as there were irregular time gaps between the registrations that were inherent to the system for this application. Data from two experiments were used: one used behavioural observations of six pigs during three days with the aim of finding the best method for visit construction (experiment 2, E2), whilst the other experiment used feeding pattern observations of 20 pigs on one day as an independent validation data-set (experiment 1, E1). Three methods for visit construction were proposed: (1) visit criteria (a bout criterion and a minimum duration criterion), (2) a digital filter (using a well-defined time delay and a threshold for feeding) and (3) erosion and dilation operations from mathematical morphology. The best results were obtained using a bout criterion equal to 10 s for two RFID tags per pig or 20 s for one RFID tag per pig. Removal of short visits was found to be unnecessary. This optimal method is equal to a closing operation (a dilation followed by an erosion). For the RFID system with the optimal bout criterion, using two tags per pig, average sensitivity was 83%, specificity 98%, accuracy 97% and precision was 75% for E2. Applying the same bout criterion on the independent test set (E1), resulted in an average sensitivity of 80%, a specificity of 99%, an accuracy of 98% and a precision of 78%. Performance of the method tended to be lower when using one tag per pig. Regression analysis revealed that the observed feeding duration was well-correlated with RFID-based feeding duration (R 2 = 0.86 for two tags per pig, E1 + E2). Other observed variables (number of feeding visits and inter-visit interval) were harder to predict by the RFID system. From these results, there was concluded that feeding patterns can be recorded accurately in an automatic way if the registrations from the RFID system are analysed using an appropriate bout criterion. [ABSTRACT FROM AUTHOR]

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. [ABSTRACT FROM AUTHOR]

Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT- 126 and the D3 domain-cluster II complex with TMT-127. Nano- LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys256, Lys270 and Lys305-Lys306 suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys256 and Lys270 are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K256A, K305A and K306A now showed that Lys306 contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys60, Lys191, Lys256, Lys270 and Lys305-Lys306 exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys60 has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys191 of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K191A, K256A, K305A and K306A, however, revealed a modest reduction in cluster II binding for the K256A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation. [ABSTRACT FROM AUTHOR]

A 3D imaging technique using a high speed binocular stereovision system was developed in combination with corresponding image processing algorithms for accurate determination of the parameters of particles leaving the spinning disks of centrifugal fertilizer spreaders. Validation of the stereo-matching algorithm using a virtual 3D stereovision simulator indicated an error of less than 2 pixels for 90% of the particles. The setup was validated using the cylindrical spread pattern of an experimental spreader. A 2D correlation coefficient of 90% and a Relative Error of 27% was found between the experimental results and the (simulated) spread pattern obtained with the developed setup. In combination with a ballistic flight model, the developed image acquisition and processing algorithms can enable fast determination and evaluation of the spread pattern which can be used as a tool for spreader design and precise machine calibration. [ABSTRACT FROM AUTHOR]

Background/Aims: Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants) in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs). Methods: Cultured human umbilical venous endothelial cells (HUVECs) and glomerular microvascular endothelial cells (GMVECs) were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF) in the supernatant as well as the remaining intracellular fraction was determined. Results: In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm2 resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm2, no effect in GMVECs was observed. Conclusion: Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP).We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the twoC1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces amotif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressingU87-MGcells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology. [ABSTRACT FROM AUTHOR]

A recent chemical foot printing study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability. [ABSTRACT FROM AUTHOR]

Lameness in sows is an important welfare concern as well as a cause of considerable economic loss. In general, lameness in sows is evaluated by visual scoring of the gait. However, visual assessment is a subjective technique with low reproducibility. Therefore, the need arises for more accurate and objective methods to enable correct detection. A detection system, based on force stance variables derived from force plate analysis and visual stance variables derived from image processing was developed: SowSIS (sow stance information system). The transportable device and computer software was tested in a practical environment and was positively evaluated for its practical use and precise measurements. A preliminary study of the force stance variables was performed by comparing measurements of sound sows and sows with lameness in their hind leg. All lame sows were reluctant to bear weight on their lame leg which resulted in a lower weight exerted by the lame hind leg compared to the other legs. This shift in weight was mainly compensated by putting more weight on the contralateral sound hind leg. Sows showed more but shorter kicks (i.e. lifting leg of the ground) with their lame leg compared to the other legs. The results indicated that the developed system SowSIS has very promising potential to support future objective research in lameness in sows. [ABSTRACT FROM AUTHOR]

Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser12B5Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Glnor p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona tide cell model to study the pathogenic effects Of VWF mutations. [ABSTRACT FROM AUTHOR]

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser- 766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and theα-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser- 764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WTVWF. The results of our study show that the Nterminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism. [ABSTRACT FROM AUTHOR]

Wideband receiver (RX) and transmitter (TX) RF front-ends for wireless universal radio applications are presented. The RX is comprised of a two-stage low-noise amplifier (LNA) applying feedback and shunt peaking, a combiner buffer for performance boosting, and an inverter-based in-phase/quadrature (IQ) down-conversion mixer. The wideband LNA provides input matching of better than -\ 10 dB from dc to beyond 10 GHz. The conversion gain (CG) of the RX front-end has a peak value of 31 dB with a 3-dB bandwidth up to 9 GHz. The minimal noise figure is 6 dB and kept below 9 dB within the entire operational bandwidth. The RX has a linearity in terms of intermodulation distortion of better than -\12 dBm. The direct conversion TX involving inverter-based IQ modulator and Darlington-type pre-power amplifier features operation up to 9 GHz with 10-dB mean CG and an average output power of 4 dBm at 1-dB compression level. Excluding buffers and local oscillator generation, the RX and TX dissipate 54 and 84 mW, respectively, from a 1.2-V voltage supply. The circuit prototypes have been fabricated in a standard 65-nm CMOS low-power process without any additional RF options and occupy an area of only 0.77 \ mm^2 and 0.53 \ mm^2, respectively. [ABSTRACT FROM PUBLISHER]

Galectin-8 (Gal8) interacts with β-galactoside-containing glycoproteins and has recently been implicated to play a role in platelet activation. It has been suggested that Gal8 may also interact with platelet coagulation factor V (FV). This indispensable cofactor is stored in a-granules of platelets via a poorly understood endocytic mechanism that only exists in megakaryocytes (platelet precursor cells). In this study, we now assessed the putative role of Gal8 for FV biology. Surface plasmon resonance analysis and a solid phase binding assay revealed that Gal8 binds FV. The data further show that β-galactosides block the interaction between FV and Gal8. These findings indicate that Gal8 specifically interacts with FV in a carbohydrate-dependent manner. Confocal microscopy studies and flow cytometry analysis demonstrated that megakaryocytic DAMI cells internalize FV. Flow cytometry showed that these cells express Gal8 on their cell surface. Reducing the functional presence of Gal8 on the cells either by an anti-Gal8 antibody or by siRNA technology markedly impaired the endocytic uptake of FV. Compatible with the apparent role of Gal8 for FV uptake, endocytosis of FV was also affected in the presence of β-galactosides. Strikingly, thrombopoietin-differentiated DAMI cells, which represent a more mature megakaryocytic state, not only lose the capacity to express cell-surface bound Gal8 but also lose the ability to internalize FV. Collectively, our data reveal a novel role for the tandem repeat Gal8 in promoting FV endocytosis. [ABSTRACT FROM AUTHOR]

As apoptosis occurs over an interval of time after administration of apoptosis-inducing therapy in tumors, the changes in technetium 99m (99mTc)-tricarbonyl (CO)3 His-annexin A5 (His-ann A5) accumulation over time were examined. Colo205-bearing mice were divided into six treatment groups: (1) control, (2) 5-fluorouracil (5-FU; 250 mg/kg), (3) irinotecan (100 mg/kg), (4) oxaliplatin (30 mg/kg), (5) bevacizumab (5 mg/kg), and (6) panitumumab (6 mg/kg). 99mTc-(CO)3 His-ann A5 was injected 4, 8, 12, 24, and 48 hours posttreatment, and micro--single-photon emission computed tomography was performed. Immunostaining of caspase-3 (apoptosis), survivin (antiapoptosis), and LC3-II (autophagy marker) was also performed. Different dynamics of 99mTc-(CO)3 His-ann A5 uptake were observed in this colorectal cancer xenograft model, in response to a single dose of three different chemotherapeutics (5-FU, irinotecan, and oxaliplatin). Bevacizumab-treated mice showed no increased uptake of the radiotracer, and a peak of 99mTc-(CO)3 His-ann A5 uptake in panitumumab-treated mice was observed 24 hours posttreatment, as confirmed by caspase-3 immunostaining. For irinotecan-, oxaliplatin-, and bevacizumab-treated tumors, a significant correlation was established between the radiotracer uptake and caspase-3 immunostaining (r = .8, p < .05; r = .9, p < .001; r = .9, p < .001, respectively). For 5-FU- and panitumumab-treated mice, the correlation coefficients were r = .7 (p = .18) and r = .7 (p = .19), respectively. Optimal timing of annexin A5 imaging after the start of different treatments in the Colo205 model was determined. [ABSTRACT FROM AUTHOR]

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys1967 and Lys1968 of region Thr1964-Tyr1971 are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys1967-Lys1968 within region Thr1964- Tyr1971 has an opposite contribution to the stability of FVIIIa. [ABSTRACT FROM AUTHOR]

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings. [ABSTRACT FROM AUTHOR]

Cattle lameness causes considerable animal welfare problems and negatively affects the farm economy. Gait scoring techniques and claw health reports are commonly used for research and surveys, but few daily management solutions exist to monitor gait parameters from individual cows within a herd. A tool to automate and process the measurement of spatiotemporal kinematic and force variables was developed using a pressure sensitive walkway, commercial farm infrastructure and management tools. A fully automatic setup on ILVO’s experimental farm measures and analyses the gait of each cow that exits the milking parlour by registration of 20 basic kinematic gait variables. Based on this variable set, a wide range of typical gait parameters such as triple support time, abduction, etc. can be calculated. This paper presents some practical results and considerations related to this system of automated gait analysis. A first validation of the acquired variables shows that the Gaitwise system is capable of clustering observations in relation to the observer score with an overall sensitivity of 76–90% and specificity of 86–100%. Variables of asymmetry and speed seem most promising for further research on the detection of lameness. For future reference, the resulting cow gait variables will be stored for time series analysis to detect changes in individual cow walking behaviour in relation to any source (claw lesions, gestation stage, etc.). Such measurements could provide valuable information for management, veterinary check-ups and further research for automatic lameness detection in cattle. [ABSTRACT FROM AUTHOR]

Abstract: Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092–2093 of the C1 domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092–2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092–2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding. [Copyright &y& Elsevier]

Background: Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood. Methodology/Principal Findings: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH. Conclusions: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by complement-type repeats (CR) each comprising a binding pocket for a single basic amino acid residue. It has been proposed that at least two CRs are required for high-affinity interaction by utilising two spatially distinct lysine residues on the ligand surface. LDL receptor-related protein (LRP) mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We now addressed the role of the two critical lysines not only in LRP binding but also in LRP-dependent endocytosis. Variants of the third domain (D3) of receptor-associated protein (RAP) were created carrying lysine to alanine or arginine replacements at the putative contact residues K253, K256 and K270. Surface plasmon resonance revealed that replacement of K253 did not affect high-affinity LRP binding at all, whereas replacement of either K256 or K270 markedly reduced the affinity by approximately 10-fold. Binding was abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding. This suggests that despite their positive charge, arginine residues do not support receptor binding at all. Confocal microscopy and flow cytometry studies surprisingly revealed that the single mutants were still taken up and still competed for the uptake of full length RAP despite their receptor binding defect. We therefore propose that the presence of only one of the two critical lysines is sufficient to drive endocytosis. [Copyright &y& Elsevier]

Abstract: The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated α2-macroglobulin (α2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3–10) and cluster IV of 11 repeats (CR21–31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding α2M* and RAP. We now evaluated the binding to RAP, α2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4–8 of cluster II and within region CR24–28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5–6, CR26–27), combinations of three repeats showed most efficient binding to FIXa (CR6–8, CR26–28), FVIII light chain (CR5–7, CR6–8, CR24–26), and α2M* (CR4–6, CR24–26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands. [Copyright &y& Elsevier]

Low-density lipoprotein receptor-related protein (LRP) contributes to factor VIII (FVIII) catabolism in vivo. Besides LRP, FVIII also interacts with very low-density lipoprotein receptor (VLDLR) in vitro. We investigated the physiological role of VLDLR in FVIII catabolism, using knockout mouse models for VLDLR and LRP, alone and in combination. VLDLR−/− mice displayed normal plasma FVIII, whereas VLDLR−/− LRP− double-knockout mice had slightly increased FVIII compared with LRP-deficient mice. Remarkably, VLDLR deficiency slightly accelerated FVIII clearance. Adenovirus-mediated overexpression of VLDLR did not lower plasma FVIII in LRP-deficient mice. We conclude that VLDLR does not act in concert with LRP in FVIII clearance in vivo. [ABSTRACT FROM AUTHOR]

Studies the activation of blood coagulation factor IX to activated factor IX. Development of enzymatic activity; Exposure of the binding sites; Use of an affinity-purified soluble form of lipoprotein receptor-related protein.

Focuses on the binding of multiple distinct ligands by the low density lipoprotein receptor-related protein (LRP). Interaction of light and heavy chains with LRP; Affinity of LRP; Use of synthetic peptides.'

Presents the use of Complementary Metal Oxide Semiconductors (CMOS) for integration of a Radio Frequency (RF) transceiver front-end. Attempts of producing single-chip CMOS RF circuits for cellular applications; Discussion of fundamental pitfalls and limitations of RF CMOS; Emphasis on optimizing transceiver as a single block.

The functional defect caused by substitution of Arg527 (→ Trp) and Arg531 (→ Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527. [ABSTRACT FROM AUTHOR]

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region LysT13-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X, phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3±0.3×106 M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713–1637) factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713–1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713–1637)-factor VIII is clearly reduced. Furthermore, the second-order rate constant (0.2±0.1×106 M-1 s-1) of des-(713–1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713–1637)-factor VIII light chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des (713–1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis. [ABSTRACT FROM AUTHOR]