Count-dependent filter for smoothing bivariate FCM histograms

A data-smoothing filter has been developed that permits the improvement in accuracy of individual elements of a bivariate flow cytometry (FCM) histogram by making use of data from adjacent elements, a knowledge of the two-dimensional measurement system point spread function (PSF), and the local count density. For FCM data, the PSF is assumed to be a set of two-dimensional Gaussian functions with a constant coefficient of variation for each axis. A set of space variant smoothing kernels are developed from the basic PSF by adjusting the orthogonal standard deviations of each Gaussian smoothing kernel according to the local count density. This adjustment in kernel size matches the degree of smoothing to the local reliability of the data. When the count density is high, a small kernel is sufficient. When the density is low, however, a broader kernel should be used. The local count density is taken from a region defined by the measurement PSF. The smoothing algorithm permits the reduction in statistical fluctuations present in bivariate FCM histograms due to the low count densities often encountered in some elements. This reduction in high-frequency spatial noise aids in the visual interpretation of the data. Additionally, by making more efficient use of smaller samples, systematic errors due to system drift may be minimized.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Biomechanical measurements on scaphoid bone screws in an experimental model

A number of screws commonly used for internal fixation in scaphoid bone fractures and nonunions are compared regarding biomechanical properties and clinical applicability. The experiments were carried out on models made of ash-wood, representing a reconstruction and fixation as is performed in a cortico-cancellous inlay bone graft for scaphoid non-union. For fixation use was made of 2.7 and 3.5 AO/ASIF cortical screws respectively, 4.0 AO/ASIF cancellous screws, Herbert screws, and a newly designed screw called the three components screw (D.K.S.). The models with implanted screws were tested for bending strength, tensile strength and torsion stability. No large differences between the various screws were found regarding the measured parameters, so that a small intra-osteal implant such as the Herbert screw and the D.K.S., which can be inserted easily and which gives a certain amount of interfragmentary compression, will be sufficient for osteosynthesis of the scaphoid bone. In case an intra-osteal implant is not available a single 3.5 AO/ASIF cortical screw, inserted following lag-screw principles, is recommended.     

Generation of soluble lignin-derived compounds during degradation of barley straw in an artificial rumen reactor

Summary The supernatants of effluents from an artificial rumen reactor degrading barley straw have been shown to contain lignin-derived compounds by UV spectral characteristics and pyrolysis mass spectrometry (PYMS). Most of these compounds were shown to be released by the action of rumen microorganisms. The compounds were quantified by measuring absorbance at 280 nm using bamboo-milled wood lignin as a standard. The concentration of the compounds rose from 0.5 mg·ml^–1 at solid and liquid retention times (SRT and HRT) of 60 and 12 h, respectively, and a loading rate (LR) of 25 g total solids (TS)·l^–1 per day to 3.5 mg·ml^–1 at a SRT of 144 h, an HRT of 20 days and an LR of 15 g TS·1^–1 per day. The highest concentration was below the level known to be toxic to rumen microorganisms in vitro. No indications were found for anaerobic lignin degradation in the rumen reactor. Offprint requests to: H. J. M. Op den Camp     

Large DNA inversions,deletions, and TaqI site mutations in severe haemophilia A

Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5 of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3 region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5 and 3 sequences around the breakpoints. A novel missense mutation is also reported: CGAGGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.     

The embryonic development of the Drosophila visual system

We have used electron-microscopic studies, bromodeoxyuridine (BrdU) incorporation and antibody labeling to characterize the development of the Drosophila larval photoreceptor (or Bolwig's) organ and the optic lobe, and have investigated the role of Notch in the development of both. The optic lobe and Bolwig's organ develop by invagination from the posterior procephalic region. After cells in this region undergo four postblastoderm divisions, a total of approximately 85 cells invaginate. The optic lobe invagination loses contact with the outer surface of the embryo and forms an epithelial vesicle attached to the brain. Bolwig's organ arises from the ventralmost portion of the optic lobe invagination, but does not become incorporated in the optic lobe; instead, its 12 cells remain in the head epidermis until late in embryogenesis when they move in conjunction with head involution to reach their final position alongside the pharynx. Early, before head involution, the cells of Bolwig's organ form a superficial group of 7 cells arranged in a rosette pattern and a deep group of 5 cells. Later, all neurons move out of the surface epithelium. Unlike adult photoreceptors, they do not form rhabdomeres; instead, they produce multiple, branched processes, which presumably carry the photopigment. Notch is essential for two aspects of the early development of the visual system. First, it delimits the number of cells incorporated into Bolwig's organ. Second, it is required for the maintenance of the epithelial character of the optic lobe cells during and after its invagination.