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In support of the (pre-print) publication "Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells" we share the following datasets: SEAP activity and calibration curve values ELISA values Flow cytometry FCS files raw blot and gel images raw grayscale fluorescence microscopy images

The tremendous recent developments in click chemistry, including the impressive developments of strain-promoted cycloaddition reagents, all started with the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction conceived by Meldal et al. and Sharpless et al. This led to a revolution of extremely important applications in the chemical, biological, medical, and materials sciences. It is fair to state that, especially in the synthesis of multifunctional and complex small-to-large biomolecular constructs, CuAAC has been indispensable. This has been particularly evident in the area of peptides, peptidomimetics, and protein mimics. These biomolecules play key roles in the various peptide–peptide, peptide–protein, and protein–protein interactions that are involved in many diseases and disorders, and peptide-based therapeutics can be important in this context. However, it is often important to improve the bioactivity and overall stability, and modulate the spatial structure, of peptide-based therapeutics. The incorporation of the 1,4-disubstituted 1,2,3-triazole moiety as a non-native structural element using CuAAC is explored in this chapter. The resulting incorporated triazole moiety can lead to structural surrogates of the amide bond and disulfide bond. As a consequence, CuAAC can be utilized toward introducing conformational constraints and stabilizing secondary structures of α-helices, β-sheets/turns, or loop-like structures. In addition, CuAAC can be used to combine various peptide sequences with molecular scaffolds to develop protein mimics that can find applications as synthetic vaccines and antibodies.

Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.

In this research we describe the improvement of the water-solubility of cyclic epitope mimics based on the HCV E2 glycoprotein by incorporation of suitable polar hinges. The poor solubility of epitope mimics based on peptide sequences in the envelope (E2) protein hampered their synthesis and purification and made it very difficult to prepare the molecular constructs for evaluation of their bioactivity. Since changes in the amino acid composition are hardly possible in these epitope mimics in order to increase water-solubility, a polar cyclization hinge may offer a remedy leading to a significant increase of polarity and therefore water solubility. These polar hinges were applied in the synthesis of better water-soluble HCV-E2 epitopes. An azide functionality in the polar hinges allowed attachment of a tetraethylene glycol linker by Cu-catalyzed azide-alkyne cyclo-addition (CuAAC) for a convenient conjugation to ELISA plates in order to evaluate the bio-activity of the epitope mimics. The immunoassays showed that the use of more polar cyclization hinges still supported anti-HCV antibody recognition and did not negatively influence their binding. This significantly increased solubility induced by polar hinges should therefore allow for the molecular construction and ultimate evaluation of synthetic vaccine molecules.

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e., epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate surfaces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To the best of our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity toward their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key binding residues by simply changing the amino acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key binding residue for binding monoclonal antibody V3.2.

The objectives of this study were to determine the reflectance properties of volunteer potato and sugar beet and to assess the potential of separating sugar beet and volunteer potato at different fields and in different years, using spectral reflectance characteristics. With the ImspectorMobile, vegetation reflection spectra were successfully repeatedly gathered in two fields, on seven days in 2 years that resulted in 11 datasets. Both in the visible and in the near-infrared reflection region, combinations of wavelengths were responsible for discrimination between sugar beet and volunteer potato plants. Two feature selection methods, discriminant analysis (DA) and neural network (NN), succeeded in selecting sets of discriminative wavebands, both for the range of 450–900 and 900–1650 nm. First, 10 optimal wavebands were selected for each of the 11 available datasets individually. Second, by calculating the discriminative power of each selected waveband, 10 fixed wavebands were selected for all 11 datasets analyses. Third, 3 fixed wavebands were determined for all 11 datasets. These three wavebands were chosen because these had been selected by both DA and NN and were for sensor 1: 450, 765, and 855 nm and for sensor 2: 900, 1440, and 1530 nm. With the resulting three sets of wavebands, classifications were performed with a DA, a neural network with 1 hidden neuron (NN1) and a neural network with two hidden neurons (NN2). The maximum classification performance was obtained with the near-infrared sensor coupled to the NN2 method with an optimal adapted set of 10 wavebands, where the percentages were 100 ± 0.1 and 1 ± 1.3% for true negative (TN) classified volunteer potato plants and false negative (FN) classified sugar beet plants respectively. In general the NN2 method gave the best classification results, followed by DA and finally the NN1 method. When the optimal adapted waveband sets were generalized to a set of 10 fixed wavebands, the classification results were still at a reasonable level of a performance at 87% TN and 1% FN for the NN2 classification method. However, when a further reduction and generalization was made to 3 fixed wavebands, the classification results were poor with a minimum performance of 69% TN and 3% FN for the NN2 classification method. So, these results indicate that for the best classification results it is required that the sensor and classification system adapt to the specific field situation, to optimally discriminate between volunteer potato and sugar beet pixel spectra

The paper presents results of research on inverse kinematics algorithms to be used in a functional model of a cucumber harvesting robot consisting of a redundant manipulator with one prismatic and six rotational joints (P6R). Within a first generic approach, the inverse kinematics problem was reformulated as a non-linear programming problem and solved with a generic algorithm. Solutions were easily obtained, but the considerable calculation time needed to solve the problem prevented on line implementation. To circumvent this problem, a second, less generic approach was developed consisting of a mixed numerical-analytic solution of the inverse kinematics problem exploiting the particular structure of the P6R manipulator. This approach facilitated rapid and robust calculation of the inverse kinematics of the cucumber harvester. During the early stages of the cucumber harvesting project, this inverse kinematics algorithm was used to off-line evaluate the ability of the robot to harvest cucumbers using 3D-information of a cucumber crop obtained in a real greenhouse. Thereafter, the algorithm was employed successfully in a functional model of the cucumber harvester to determine if cucumbers were hanging within the reachable workspace of the robot and to determine a collision-free harvest posture to be used for motion control of the manipulator during harvesting.

Mimics of discontinuous epitopes of for example bacterial or viral proteins may have considerable potential for the development of synthetic vaccines, especially if conserved epitopes can be mimicked. However, due to the structural complexity and size of discontinuous epitopes molecular construction of these mimics remains challeging. We present here a convergent route for the assembly of discontinuous epitope mimics by successive azide alkyne cycloaddition on an orthogonal alkyne functionalized scaffold. Here the synthesis of mimics of the HIV gp120 discontinuous epitope that interacts with the CD4 receptor is described. The resulting protein mimics are capable of inhibition of the gp120-CD4 interaction. The route is convergent, robust and should be applicable to other discontinuous epitopes.

Grassland management has a large influence on the operating cost and environmental impact of dairy farms and requires accurate, detailed, and timely information about the yield and quality of grass. Our objective was to evaluate imaging spectroscopy as a means to obtain accurate, detailed, and rapid measurements of grass yield and quality. The work consisted of three steps. In the first step, a new mobile measurement system comprising several hyperspectral sensors was constructed and calibrated on measurements collected in six field experiments in the Netherlands in 2 yr. A partial least squares regression model was used to fit parameters derived from hyperspectral images to values of DM (dry matter) yield and quality obtained through destructive sampling. Leave-k-out cross validation showed relative errors of prediction of 8 to 22% (167¿477 kg DM ha¿1 absolute) for DM yield, 21% (0.07 absolute) for the fraction of clover in DM, 6 to 12% for nutrient concentration, 15 to 16% for sugar concentration, and 3 to 5% for feeding values. In the second step, the measurement system was used to predict grassland yield and quality on fields from two farms. In the third step, the absence of calibration data for a specific date was simulated with a leave-harvest-out procedure. Predictions of absolute values were strongly biased due to system instability. Prediction of relative values was good, with a mean absolute error of 183 kg ha¿1 for DM yield. The instability of the measurement system requires that duosampling must be used for prediction of absolute values.

Hereditary neuralgic amyotrophy (HNA) is an autosomal-dominant inherited recurrent focal neuropathy affecting mainly the brachial plexus. In this study we report the genomic structure and mutation analysis of three candidate genes: sphingosine kinase 1 (SPHK1); tissue inhibitor of metalloproteinase 2 (TIMP2); and cytoglobin (CYGB). We did not find any disease-associated mutations, indicating that HNA is not caused by point mutations in these genes. However, we identified several sequencing errors in the cDNA of SPHK1 as well as seven novel single-nucleotide polymorphisms.

Attempts to improve grassland management may benefit from the use of new sensing techniques, such as imaging spectroscopy. In order to explore the potential of hyperspectral imaging spectroscopy for rapid and objective characterization of grass swards an experimental prototype has been developed. From a height of 1 m, the system detects reflections at high spatial (0·16–1·45 mm2) and high spectral resolutions (5–17 nm) at wavelengths between 405 and 1659 nm. Detailed information is provided on design, characteristics and test results. Results show that reflection intensity is related to height position in the sward and leaf angle. Grass leaves were recognized accurately. Images of a sports field and a production sward with similar ground cover fractions could be easily distinguished. With the described system, canopy structure can be characterized by ground cover, the distribution of reflection intensity and image texture. Spectral characteristics can be obtained at leaf level.

This paper describes the concept of an autonomous robot for harvesting cucumbers in greenhouses. A description is given of the working environment of the robot and the logistics of harvesting. It is stated that for a 2 ha Dutch nursery, 4 harvesting robots and one docking station are needed during the peak season. Based on these preliminaries, the design specifications of the harvest robot are defined. The main requirement is that a single harvest operation may take at most 10 s. Then, the paper focuses on the individual hardware and software components of the robot. These include, the autonomous vehicle, the manipulator, the end-effector, the two computer vision systems for detection and 3D imaging of the fruit and the environment and, finally, a control scheme that generates collision-free motions for the manipulator during harvesting. The manipulator has seven degrees-of-freedom (DOF). This is sufficient for the harvesting task. The end-effector is designed such that it handles the soft fruit without loss of quality. The thermal cutting device included in the end-effector prevents the spreading of viruses through the greenhouse. The computer vision system is able to detect more than 95% of the cucumbers in a greenhouse. Using geometric models the ripeness of the cucumbers is determined. A motion planner based on the Aa-search algorithm assures collision-free eye-hand co-ordination. In autumn 2001 system integration took place and the harvesting robot was tested in a greenhouse. With a success rate of 80%, field tests confirmed the ability of the robot to pick cucumbers without human interference. On average the robot needed 45 s to pick one cucumber. Future research focuses on hardware and software solutions to improve the picking speed and accuracy of the eye-hand co-ordination of the robot.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating peripheral neuropathy. Clinical hallmarks are recurrent painless focal neuropathies mostly preceded by minor trauma or compression at entrapment sites of peripheral nerves. In the majority of the patients, HNPP is caused by a 1.5 Mb deletion on chromosome 17p11.2-p12 containing the peripheral myelin protein 22 (PMP22) gene. Point mutations within this gene are reported in only a few families. We report a novel mutation in the PMP22 gene in a Spanish family with HNPP. The mutation is a 3′ splice-site mutation, preceding coding exon 3 (c.179-1 G>C), causing a mild HNPP phenotype.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.

HNA is an autosomal dominant recurrent focal neuropathy involving the brachial plexus. The etiology of HNA is unknown but the genetic defect most likely affects a non-neuronal tissue. We previously described linkage to chromosome 17q24-q25 in two HNA-families. Here we report the mutation analysis of two candidate genes: a cDNA encoding a putative sialyltransferase and the SFRS2 splicing factor including the c-myb ET-locus which is encoded on the opposite strand of the SFRS2 gene. The complete protein coding regions of both genes were studied by direct DNA sequencing. We did not find a disease associated mutation indicating that these genes are most likely not involved in the pathogenesis of HNA. However, we identified and characterized a rare AvaII polymorphism in the SFRS2 gene and detected a sequencing error, leading to an amino acid change (Val11Leu) in the published sequence of the putative sialyltransferase.

Digital image processing (DIP) is a potential tool for measuring and classifying pot plants in various growth stages in an automatic, objective and consistent way, with a high capacity and low labour input. In this study, features of begonia cuttings which could be relevant for grading were analysed. Images of unrooted and rooted Begonia elatior [B. hiemalis] cuttings were acquired and analysed with DIP. The various parts of the cuttings were identified and measured using knowledge based image processing. These measurements were shown to be consistent and to be well correlated with the features measured in conventional ways. Experts graded the rooted cuttings into 3 classes: small, medium and large. The effect of grading unrooted cuttings and growing them with similar-sized cuttings was still apparent 4 weeks later - the rooted cuttings in graded units were more uniform than those in random units. Two models were constructed to determine the quality of the rooted cuttings based on DIP measurements, one based on multiple linear regression and one based on a neural network. Both were able to grade at least 75% of the rooted cuttings in the same class as the expert. The neural network-based model performed slightly (5%) better, especially for the classification of small and large plants. The lack of objective quality criteria is a major obstacle for the development of grading models for pot plants.

Upper limb postural tremor consists of mechanical-reflex and central-neurogenic oscillations, superimposed upon a background of irregular fluctuations in muscle force. Muscle spindles play key-roles in the information flow to supra-spinal and spinal generators. Oscillations were delivered using a new generation portable myohaptic device, called ldquowristalyzer,rdquo taking into account the ergonomy of upper limbs and allowing a fine adjustment to each configuration of upper limb segments. The nominal torque of the first generation device is 4 Nm, with a maximal rotation velocity of 300 degrees/s and a range of motion of plusmn45 degrees. Reliability was assessed in basal condition and during loading conditions. We assessed the effects of the addition of inertia on postural tremor of the finger in a group of 26 neurological patients and the effects of wrist oscillations upon contralateral postural tremor in 6 control subjects and in 7 neurological patients exhibiting a postural tremor. Patients showed two different behaviors in response to inertia and exhibited an increased variability of postural tremor during fast oscillations (13.3 Hz). One patient with overactivity of the olivocerebellar pathways exhibited a drop in the peak frequency of more than 20%. The relative power of the 8-12 Hz subband was significantly higher in controls both in basal condition and during oscillations (p = 0.028 and p = 0.015, respectively). The second generation wristalyzer allows to investigate the effects of mechanical oscillations up to frequency of 50 Hz. This mechatronic device can assess the responsiveness of tremor generators to stimulation of muscle spindles and biomechanical loading. Potential applications are the monitoring of dysmetria under various inertial or damping conditions, the assessment of rigidity in Parkinson's disease and the characterization of voluntary muscle force.

The formability of a material depends upon the strain hardening and strain rate hardening of the material. In this study, constitutive parameters using the power law constitutive equation are determined for six different strength steels and two aluminum alloys over different strain ranges, including approximations of the postuniform elongation range. Constitutive parameters are found to be different at different strain ranges. The strain hardening of steels increases with strain at low strain levels (less than 5%) and decreases at high strain levels (greater than 10%). Strain rate hardening decreases with strain for all steels and aluminum alloys. Uniform elongation depends only on strain hardening, and postuniform elongation depends only on strain rate hardening. However, the total elongation depends on both strain hardening and strain rate hardening.

The paper presents results of research on an inverse kinematics algorithm that has been used in a functional model of a cucumber-harvesting robot consisting of a redundant P6R manipulator. Within a first generic approach, the inverse kinematics problem was reformulated as a non-linear programming problem and solved with a Genetic Algorithm (GA). Although solutions were easily obtained, the considerable calculation time needed to solve the problem prevented on-line implementation. To circumvent this problem, a second, less generic, approach was developed which consisted of a mixed numerical-analytic solution of the inverse kinematics problem exploiting the particular structure of the P6R manipulator. Using the latter approach, calculation time was considerably reduced. During the early stages of the cucumber-harvesting project, this inverse kinematics algorithm was used off-line to evaluate the ability of the robot to harvest cucumbers using 3D-information obtained from a cucumber crop in a real greenhouse. Thereafter, the algorithm was employed successfully in a functional model of the cucumber harvester to determine if cucumbers were hanging within the reachable workspace of the robot and to determine a collision-free harvest posture to be used for motion control of the manipulator during harvesting. The inverse kinematics algorithm is presented and demonstrated with some illustrative examples of cucumber harvesting, both off-line during the design phase as well as on-line during a field test.

In 2006 the project ISAFRUIT (www.isafruit.org)--"Increasing fruit consumption through a trans disciplinary approach leading to high quality produce from environmentally safe, sustainable methods"--was launched within the 6th Framework Program of the EC. Within the project's work package ECOFRUIT (WP 5.1 - Safe European fruit from a healthy environment) a Crop Adapted Spray Application (CASA) system is developed. This system is developed to ensure efficient and safe spray application in orchards according to actual needs and with respect to the environment. The system consists of three components: 1. Crop Identification System (CIS), 2. Environmentally Dependent Application System (EDAS), and 3. Crop Health Sensor (CHS). A sprayer prototype able to automatically adapt spray and air distribution according to the characteristics of the target, to the level of crop disease and to the environmental conditions is under development.

We investigated fast pointing and fast reversal single-joint movements performed under artificial damping conditions applied with the myohaptic technology. Healthy subjects were able to scale the intensity of the agonist and antagonist electromyographic (EMG) activities when damping was added during both types of movements. Cerebellar patients were able to scale the magnitude of the agonist and antagonist EMG activities during pointing movements. For reversal movements, patients could scale the intensity of the initial agonist EMG activity but were unable to modulate the intensities of the second set of EMG discharges associated with the return to the initial position. Patients were able to adopt the appropriate motor strategy for the first phase of reversal movements, but they were unable to implement the suitable sequential strategy for the superimposition of motor plans during artificial damping. The myohaptic technology is the first to allow the detection of deficits in the implementation of sequential motor coding under the mechanical condition of artificial damping.

We report a novel form of hypometria in a patient presenting a spinocerebellar ataxia type 2 (SCA2; 32 repeats; Age: 44). We investigated fast pointing (FP) and fast reversal (FR) single-joint movements performed under artificial damping conditions using our myohaptic device called Wristalyzer, which allows an instantaneous regulation of the mechanical characteristics of a wrist manipulandum. The wristalyzer controller has a range of motion from −1 rad to +1 rad. The inertia of the motor/handgrip is 0.004 kg × m2. Movements were studied in free mode and with damping either at 0.1 Nms/rad or 0.2 Nms/rad. Subjects performed sets of FP movements and FR movements over 3 distances (targets: 0.2, 0.3, 0.4 rad). Control subjects (n = 8, mean age: 34.8 +/− 10.2 years; 3 women) were able to scale the intensity of the agonist and antagonist electromyographic (EMG) activities when damping was added during both types of movements. FR movements were accurate in the 3 mechanical conditions: mean movement amplitude was −0.0179 rad, 0.0061 rad and 0.0062 rad in the basal state, following addition of 0.1 Nms/rad and following addition of 0.2 Nms/rad, respectively. The patient scaled the magnitude of the agonist and antagonist EMG activities during FP, but FR movements were dysmetric. The second phase of reversal movements was hypometric. Movement performed in the basal condition was accurate during phase 1 but was hypometric during phase 2. The hypometria worsened with damping: mean movement amplitude (second zero-crossing of the velocity curve) was 0.088 rad in the basal state, 0.1425 rad following addition of 0.1 Nms/rad, and 0.2003 rad with addition of a damping of + 0.2 Nms/rad. This is the first demonstration in human of a deficit in the implementation of the suitable sequential strategy for the superposition of motor plans during artificial damping.

Wristalyzer is a portable robotic device combining haptic technology with electromyographic assessment. It allows to assess wrist motion in physiological and pathological conditions by applying loads and mechanical oscillations, taking into account the ergonomy and the angular positioning of the joints. The wristalyzer works in a free or loaded mode for assessment of metrics of motion and tremor, analyzes the behavior of the wrist joints and the associated muscle activities during delivery of mechanical oscillations, estimates the maximal voluntary contraction, assesses automatically the impedance of the wrist for assessment of rigidity or spasticity. Position, torques and electromyographic activities are analyzed in real time. The device characterizes the effects of damping on voluntary motion. A personal computer implements control loops and user application. This is the first standardized tool to assess wrist motion with high accuracy and reliability using the haptic technology with concomitant investigation of muscle activity.

Two simulative forming tests for assessing the adhesion of metallic coatings on sheet steel, the double reverse Olsen adhesion test and a new test, the stretch-draw adhesion test, are contrasted. The strain states, the nature of the test results, and the responses of various hot dip galvanized and electroplated zinc and zinc alloy coatings are compared. The quantitative mass loss measurements of both tests are shown to have greater relevance than the qualitative visual rating system most commonly used with the double reverse Olsen test. Both tests effectively differentiate good and poor adhesion of alloy coatings. Only small amounts of zinc coatings are lost in both tests. The double reverse Olsen test is easier to perform but represents a sequence of strain states seldom encountered in actual stamping operations.

Hereditary neuralgic amyotrophy is a rare autosomal dominant disorder of the peripheral nervous system. Previous segregation analysis in two large pedigrees suggested linkage to distal 17q. Linkage data obtained in the present study investigating a three generation pedigree confirm linkage to 17q24-q25.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy affecting the brachial plexus. HNA is triggered by environmental factors such as infection or parturition. We report three mutations in the gene septin 9 (SEPT9) in six families with HNA linked to chromosome 17q25. HNA is the first monogenetic disease caused by mutations in a gene of the septin family. Septins are implicated in formation of the cytoskeleton, cell division and tumorigenesis.

To evaluate the results of intracytoplasmatic sperm injection (ICSI) with surgically retrieved epididymal semen.Prospective, descriptive.Patients with an obstructive azoospermia confirmed by cytological examination of a testis biopsy, and conforming to the regular IVF/ICSI criteria as laid down in 2001 at the University Medical Centre St Radboud Nijmegen, the Netherlands, were included for percutaneous epididymal sperm aspiration (PESA) and ICSI. The main outcome measure was the ongoing pregnancy rate per initiated cycle.A total of 44 cycles were started in 31 couples. In 43 cases (98%) an ovum pick-up was performed and 41 (93%) embryo transfers were carried out. In 19 (43%) cases, treatment resulted in a positive pregnancy test and 15 (34%) ongoing pregnancies were recorded. In total, 17 healthy children were born (11 boys and 6 girls).ICSI with surgically retrieved semen was successfully used as a treatment for childlessness caused by obstructive azoospermia.

To evaluate diagnostic procedures and surgical sperm retrieval in men with suspected obstructive azoospermia who wish to father children.Descriptive, retrospective.During the period 1 April 1999-31 December 2001 93 men suspected of having obstructive azoospermia underwent surgical sperm retrieval by means of percutaneous epididymal sperm aspiration (PESA). In each patient a testicular biopsy was performed to determine the Johnsen score (a scoreor = 8 is equivalent to a normal spermatogenesis). Cryopreservation was performed whenever possible. The findings in both percutaneous and surgical sperm retrieval were compared.In 76 patients (82%) epididymal motile sperm were obtained using PESA. Their Johnsen score on the testis biopsy was 9.1 (range: 7.4-10). In 73 of the patients the Johnsen score wasor = 8. In the 17 patients (18%) in whom no sperm were found with PESA, the median Johnsen score was 5.8 (range: 2-9.8). Epididymal sperm were not found in patients with a testicular volume15 ml. In all 28 patients who had undergone a vasectomy in the past, motile sperm were found along with a Johnsen scoreor = 8. In 23 of the 24 patients with congenital bilateral absence of the vas deferens (CABVD) the Johnsen score wasor = 8. Cryopreservation was possible in 45 (59%) of all patients and in 5 (35%) of the 13 patients with an unknown cause for the obstructive azoospermia.In men with suspected obstructive azoospermia in whom sperm were found using PESA, a diagnostic testis biopsy provided no additional relevant information about the spermatogenesis. There was always a good spermatogenesis after vasectomy. CBAVD patients probably had at least some focal areas in the testes with normal spermatogenesis. Sperm retrieval and cryopreservation could be carried out less frequently in the case of obstructions with an unknown cause.

This paper deals with the effects of various approaches for modeling of strain rate effects for mild and high strength steels (HSS) on impact simulations. The material modeling is discussed in the context of the finite element method (FEM) modeling of progressive crush of energy absorbing automotive components. The characteristics of piecewise linear plasticity strain rate dependent material model are analyzed and various submodels for modeling of impact response of steel structures are investigated. The paper reports on the ranges of strains and strain rates that are calculated in typical FEM models for tube crush and their dependence on the material modeling approaches employed. The models are compared to the experimental results from drop tower tests.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant inherited demyelinating neuropathy typically characterized by recurrent episodes of acute painless peripheral nerve palsies often preceded by minor trauma or compression at entrapment sites. However, less classical phenotypes have been reported. A 1.5 Mb deletion in chromosome 17 p11.2 has been shown to be the genetic basis of the disease in the majority of HNPP patients. The few families without this deletion harbored a mutation in the PMP22 gene. We performed a clinical, neurophysiological and molecular genetic study of 6 Spanish HNPP families. Five families (22 individuals) showed the classical chromosome 17 p11.2 deletion and one family (3 individuals) had a novel 3'splice-site mutation in PMP22. Neurophysiological abnormalities were detected in all symptomatic (n=21) and asymptomatic (n=4) deletion or mutation carriers, even in childhood. In addition to the typical presentation we observed other phenotypes: recurrent focal short-term sensory symptoms, a progressive mononeuropathy, a Charcot-Marie-Tooth (CMT) disease-like chronic progressive polyneuropathy, a chronic sensory polyneuropathy and a chronic inflammatory demyelinating polyneuropathy. We report new or very rare phenotypesThese atypical clinical aspects and intrafamilial heterogeneity are present in families with the HNPP deletion as well as in the family with the PMP22 mutation. However, the CMT disease-like chronic polyneuropathy was more common in the PMP22 mutation family. Intrafamilial heterogeneity also seemed to be more pronounced in this kinship. Patients in this family had a mild chronic motor and sensory polyneuropathy neurophysiologically characterized by delayed distal latencies, reduced nerve conduction velocities (NCV) within the demyelinating range, mildly decreased amplitudes of motor and sensory evoked potentials and absence of conduction blocks. In contrast, patients with the common HNPP deletion, regardless of their phenotype, had a diffuse increase in distal motor latencies contrasting with moderately reduced motor NCVs, preserved sensory nerve action potentials, slowing of NCVs at the common entrapment sites and occasionally conduction blocks. In this study we confirm the clinical and molecular heterogeneity of HNPP, emphasizing the need for a mutation analysis of the PMP22 gene when the common 17p11.2 deletion is not found in clinically suspected HNPP patients. We conclude that the 3'splice-site mutation in PMP22 and the common HNPP deletion have largely the same functional consequences although some clinical and neurophysiological differences were observed.

Distal hereditary motor neuropathies (distal HMNs) are characterised by degeneration of anterior horn cells of the spinal cord resulting in muscle weakness and atrophy. Distal HMN type II is genetically linked to chromosome 12q24.3 and located within a 13 cM region flanked by markers D12S86 and D12S340. We previously excluded the human phospholipase A2 group 1B gene (PLA2G1B) as the disease causing gene. Here, we report the mutation analysis of five other candidate genes localised within the distal HMN II region: the cytoskeletal proteins paxillin (PXN) and restin (RSN); the acidic ribosomal phosphoprotein, large P0 subunit (RPLP0); a nucleoside diphosphate kinase (NME2B); and the beta 3 subunit of the voltage-gated calcium channel (CACNB3). DNA sequencing of the coding regions was performed but no disease causing mutations could be identified, hence excluding these five genes for distal HMN type II.

Contactin associated protein 1 (Caspr1/Paranodin/Neurexin IV) is an axonal transmembrane molecule mainly localised at the paranodal junction. Since molecular alterations in septate-like junctions at the paranodes might have important consequences for the function of the nerve fiber, we considered that Caspr1 could be involved in the pathogenesis of inherited peripheral neuropathies. In this study, we physically mapped the Caspr1 gene on chromosome 17q21.1 and determined its genomic structure. We performed a mutation analysis of the Caspr1 gene in a cohort of 64 unrelated patients afflicted with distinct inherited peripheral neuropathies. Since no disease causing mutations were found, we suggest that Caspr1 is probably not a common cause of inherited peripheral neuropathies.

To identify a Dutch cohort of sildenafil users and describe their baseline characteristics.Each pharmacy in The Netherlands (n = 1571) was asked to identify prospectively the first 20 sildenafil prescriptions in their pharmacy over 1 year, and to complete and return a registration form. The collected data included patient characteristics, the details of the sildenafil prescription (date, prescriber, number of prescriptions, dosing), and the use of co-medication by the patient in the year preceding the sildenafil prescription.Data were collected from 4460 sildenafil prescriptions during the year under study, relating to 3477 individual patients. Most of the cohort had cardiovascular morbidity or diabetes. Sildenafil seems to have been used by a new, previously untreated population of patients with erectile dysfunction. In addition, 69 men were identified who could have been using nitrates and sildenafil concomitantly.A cohort of patients using sildenafil was identified and characterized; they appeared to be representative of sildenafil users in The Netherlands. This cohort will be followed prospectively to evaluate the medical status (particularly cardiovascular) of the patients with time.

To describe the long-term experience with the implantation of a penile prosthesis as a last resort treatment for erectile dysfunction.Retrospective and descriptive.During the period 1986-1996, 124 men received a penile prosthesis. Basic information was obtained from medical files. Seventy-six men could be traced and were sent a questionnaire. Sixty patients and 44 partners participated in the study.The median time since the implantation was 81.3 months (range: 13-139). The chance of reoperation was 35%. Seventeen percent of the patients and 25% of the spouses regretted the decision to have the implantation. Thirty-one percent no longer used the prosthesis and 15% had never used it. Twenty percent of the patients and 31% of the partners noticed an improvement in their sexual relationship whereas 26% and 23% respectively noticed a decline. Twenty percent of the patients sometimes failed to penetrate despite the prosthesis. Nevertheless, 66% would advise implantation to someone else. Almost 50% of the men reported spontaneous tumescence during sexual arousal.After the implantation of a penile prosthesis, the reoperation rate was high, and many patients and their partners were dissatisfied with their sexual relationship despite regaining a functional erection. Implantation requires thorough sexological counselling both before and after the operation.

To assess the prevalence of erectile dysfunction (ED), and its influence on both the quality of life, and health care seeking behaviour.An age-stratified random sample was drawn from men aged 40-79 years in Boxmeer, the Netherlands (n = 1771), together with their partners. A questionnaire was mailed to the study population to collect data on ED, and the quality of life, both general and disorder related. The prevalence of ED was measured with the direct question 'Do you have problems getting an erection?'. The influence of ED on sexual function was measured with the Dutch version of the Sexual Function Inventory questionnaire.Seventy percent of all men (n = 1233) and 73% of their partners (n = 1071) responded to the questionnaire. The prevalence of ED increased with age class. To the question regarding problems to get an erection, 13% (95% confidence interval: 11-15%) of all men answered affirmatively (40-49 years: 6%; 50-59: 9%; 60-69: 22%; 70-79: 38%). All men aged 40-49 years of age with ED and 16% of men aged 70-79 with ED considered their ED as bothersome. Amongst 40 to 49 year old men with ED, 64% considered their ED as a big or rather a big problem. Amongst older men this percentage was much smaller: 50-59 years: 38%; 60-69: 37%, and 70-79: 27%. Thirty-four percent of all men with ED and 16% of their spouses were dissatisfied with their sex life. Twenty-five percent of men with ED consulted a doctor, with a mean delay of 13 months. ED bore a strong correlation with the general quality of life and comorbidity.ED was a common disorder within the population. Its prevalence was higher among older age groups, but they regarded ED as less of a problem than the younger age groups. Only a quarter of all men with ED consulted a physician.

Erectile dysfunction is a frequent condition in cardiovascular patients. Since the arrival of oral erection-supporting medication, patients want to know how safe sexual activity is in cardiovascular disease in general and during use of erection-supporting medication in particular. Sexual intercourse with a steady partner causes no more cardiovascular risk than normal daily activities such as ironing, 2 kilometers of walking without climbing, paperhanging, playing golf or gardening. The relative risk of myocardial infarction during sexual activity is not significantly higher than for healthy persons. The incidence of cardiovascular morbidity and mortality is not higher among users of sildenafil. Sildenafil is contraindicated in patients using long-acting nitrates or who may need to use short-acting nitrates, because the combination may cause a sharp fall of the blood pressure. No interactions have been observed with beta-receptor blockers, calcium antagonists, thiazide and loop diuretics and ACE inhibitors. Before prescribing a symptomatic (pharmaceutical) treatment for patients with an erection disorder, attention should be given tot the sexological, psychological and medical backgrounds of the disorder. Secondary prevention of atherosclerotic risk factors is also important: regulation of blood pressure and blood sugar level, hyperlipidaemia and obesity, as well as a change of lifestyle (giving up smoking, adapting of diet and more physical exertion). Patients with a very low cardiac capacity should be advised to refrain from treatment of the erection disorder.

In the framework of the EC-funded AIR-project Objective plant quality measurement by digital image processing taking images each three weeks during more than nine months follows the development of Ficus benjamina plants. From these images a large number of features is extracted and a relation is laid between these features and the external quality by using neural networks. A segmentation procedure for classifying the pixels into object pixel (plant) and non-object pixels (background) has to be used before feature extraction. Segmentation procedures based on thresholding depend on the specific threshold that is used, especially when the transition between object and background follows a ramp instead of a step andlor the intensity of the object andlor background is not constant for the whole image. Improved versions of MAX-MIN filters for edge enhancement are less noise sensitive than other filters for edge enhancement as for example a Laplace operator. The same feature extraction procedures are applied to images with different illumination levels and that have or have not been enhanced by improved MAX-MIN filtering. The influence of image enhancement by improved MAX-MIN-filtering on the segmentation of images and consequently on the feature values will be discussed.

Charcot-Marie-Tooth disease, the most common variant of the inherited peripheral neuropathies, has a prevalence of 1/2500. Clinical, electrophysiological, neuropathological and molecular genetic studies have demonstrated extensive heterogeneity. Currently, 30 genetic loci are known for distinct CMT types and related inherited peripheral neuropathies, while many other types have been excluded for linkage to these loci. Recent molecular genetic studies have demonstrated the involvement of 8 genes that encode proteins with very diverse functions. These include a structural protein confined to the compact myelin, a cytoskeletal protein, an adhesion molecule, a gap-junction protein, a transcription factor, a receptor for a neurotrophic factor, a phosphatase and a protein involved in signal transduction and cell cycle regulation.