Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

Semantic localization in the PCL library

The semantic localization problem in robotics consists in determining the place where a robot is located by means of semantic categories. The problem is usually addressed as a supervised classification process, where input data correspond to robot perceptions while classes to semantic categories, like kitchen or corridor. In this paper we propose a framework, implemented in the PCL library, which provides a set of valuable tools to easily develop and evaluate semantic localization systems. The implementation includes the generation of 3D global descriptors following a Bag-of-Words approach. This allows the generation of fixed-dimensionality descriptors from any type of keypoint detector and feature extractor combinations. The framework has been designed, structured and implemented to be easily extended with different keypoint detectors, feature extractors as well as classification models. The proposed framework has also been used to evaluate the performance of a set of already implemented descriptors, when used as input for a specific semantic localization system. The obtained results are discussed paying special attention to the internal parameters of the BoW descriptor generation process. Moreover, we also review the combination of some keypoint detectors with different 3D descriptor generation techniques.

Tales of the hermitage : written for the instruction and amusement of the rising generation.

Reproduction of original in the Bodleian Library.

Effects of leukapheresis protocol, cell processing and cryopreservation on the generation of monocyte-derived DC for immune therapy

Background Many clinical trials of DC-based immunotherapy involve administration of monocyte-derived DCs (Mo-DC) on multiple occasions. We aimed to determine the optimal cell processing procedures and timing (leukapheresis, RBC depletion and cryopreservation) for generation of Mo-DC for clinical purposes. Methods Leukapheresis was undertaken using a COBE Spectra. Two instrument settings were compared - the standard semi-automated software (Version 4.7) (n = 10) and the fully automated software (Version 6.0) (n = 40). Density gradient centrifugation using Ficoll, Percoll, a combination of these methods or neither for RBC depletion were compared. Outcomes (including cell yield and purity) were compared for cryopreserved unmanipulated monocytes and cryopreserved Mo-DC. Results Software Version 6.0 provided significantly better enrichment for monocytes (P

Aberrant protective force generation during neural provocation testing and the effect of treatment in patients with neurogenic cervicobrachial pain

Background: Observation of the occurrence of protective muscle activity is advocated in assessment of the peripheral nervous system by means of neural provocation tests. However, no studies have yet demonstrated abnormal force generation in a patient population. Objectives: To analyze whether aberrations in shoulder girdle-elevation force during neural tissue provocation testing for the median nerve (NTPTI) can be demonstrated, and whether possible aberrations can be normalized following cervical mobilization. Study Design: A single-blind randomized comparative controlled study. Setting: Laboratory setting annex in a manual therapy teaching practice. Participants: Twenty patients with unilateral or bilateral neurogenic cervicobrachial pain. Methods: During the NTPTI, we used a load cell and electrogoniometer to record continuously the shoulder-girdle elevation force in relation to the available range of elbow extension. Following randomization, we analyzed the immediate treatment effects of a cervical contralateral lateral glide mobilization technique (experimental group) and therapeutic ultrasound (control group). Results: On the involved side, the shoulder-girdle elevation force occur-red earlier, and the amount of force at the end of the test was substantially, though not significantly, greater than that on the uninvolved side at the corresponding range of motion. Together with a significant reduction in pain perception after cervical mobilization, a clear tendency toward normalization of the force curve could be observed, namely, a significant decrease in force generation and a delayed onset. The control group demonstrated no differences. Conclusions: Aberrations in force generation during neural, provocation testing are present in patients with neurogenic pain and can be normalized with appropriate treatment modalities.

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the crimap program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSIF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSIF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent-fashion in vitro. T cells from peg-GCSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (C) 2004 by The American Society of Hematology.

Deterministic generation of tailored-optical-coherent-state superpositions

Pulsed coherent excitation of a two-level atom strongly coupled to a resonant cavity mode will create a superposition of two coherent states of opposite amplitudes in the field. By choosing proper parameters of interaction time and pulse shape the field after the pulse will be almost disentangled from the atom and can be efficiently outcoupled through cavity decay. The fidelity of the generation approaches unity if the atom-field coupling strength is much larger than the atomic and cavity decay rates. This implies a strong difference between even and odd output photon number counts. Alternatively, the coherence of the two generated field components can be proven by phase-dependent annihilation of the generated nonclassical superposition state by a second pulse.

Modern environmental improvement pathways for the coal power generation industry in Australia

In this work we assess the pathways for environmental improvement by the coal utilization industry for power generation in Australia. In terms of resources, our findings show that coal is a long term resource of concern as coal reserves are likely to last for the next 500 years or more. However, our analysis indicates that evaporation losses of water in power generation will approach 1000 Gl (gigalitres) per year, equivalent to a consumption of half of the Australian residential population. As Australia is the second driest continent on earth, water consumption by power generators is a resource of immediate concern with regards to sustainability. We also show that coal will continue to play a major role in energy generation in Australia and, hence, there is a need to employ new technologies that can minimize environmental impacts. The major technologies to reduce impacts to air, water and soils are addressed. Of major interest, there is a major potential for developing sequestration processes in Australia, in particular by enhanced coal bed methane (ECBM) recovery at the Bowen Basin, South Sydney Basin and Gunnedah Basin. Having said that, CO2 capture technologies require further development to support any sequestration processes in order to comply with the Kyoto Protocol. Current power generation cycles are thermodynamic limited, with 35-40% efficiencies. To move to a high efficiency cycle, it is required to change technologies of which integrated gasification combined cycle plus fuel cell is the most promising, with efficiencies expected to reach 60-65%. However, risks of moving towards an unproven technology means that power generators are likely to continue to use pulverized fuel technologies, aiming at incremental efficiency improvements (business as usual). As a big picture pathway, power generators are likely to play an increasing role in regional development; in particular EcoParks and reclaiming saline water for treatment as pressures to access fresh water supplies will significantly increase.

Continuous-variable Einstein-Podolsky-Rosen paradox with traveling-wave second-harmonic generation

The Einstein-Podolsky-Rosen paradox and quantum entanglement are at the heart of quantum mechanics. Here we show that single-pass traveling-wave second-harmonic generation can be used to demonstrate both entanglement and the paradox with continuous variables that are analogous to the position and momentum of the original proposal.