Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.     

Highly-efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin

Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector-containing supernatant. We showed that infection of both human primary T cells and CD4+ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low-affinity nerve growth factor receptor (deltaLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on LNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.     

Insights into carrier recombination processes in 1.3 μmGaInNAs-based semiconductor lasers attained using high pressure

The threshold current of 1.3 μm GaInNAs lasers increases by ~30%, up to a pressure of 1 GPa compared with a decrease of ~15% for Auger-dominated InGaAsP devices, indicating that direct band-to-band Anger recombination is not important in these materials. The lasing energy varies sub-linearly with pressure, indicative of the increasing interaction of the N-level with the conduction band     

Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation

We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.     

Evidence for large monomolecular recombination contribution tothreshold current in 1.3 μm GaInNAs semiconductor lasers

The spontaneous emission, L, through a window in the substrate electrode of 1.3 μm GaInNAs MQW lasers was studied as a function of current, I, and temperature, T Close to room temperature, a characteristic temperature at threshold T,(L) T was observed as expected for band-to-band recombination in ideal quantum well devices. However, T ₀(I_th)≃T/3 indicating other processes occur. Analysis of the variation of L with I, reveals that monomolecular recombination contributes more than 50% to the total current at threshold and also that some Auger recombination may be present     

A quantitative study of radiative, Auger, and defect related recombination processes in 1.3-/spl mu/m GaInNAs-based quantum-well lasers

By measuring the spontaneous emission (SE) from normally operating /spl sim/1.3-/spl mu/m GaInNAs-GaAs-based lasers we have quantitatively determined the variation of each of the current paths present in the devices as a function of temperature from 130 K to 370 K. From the SE measurements we determine how the current I close to threshold, varies as a function of carrier density n, which enables us to separate out the main current paths corresponding to monomolecular (defect-related), radiative or Auger recombination. We find that defect-related recombination forms /spl sim/55% of the threshold current at room temperature (RT). At RT, radiative recombination accounts for /spl sim/20% of I/sub th/ with the remaining /spl sim/25% being due to nonradiative Auger recombination. Theoretical calculations of the threshold carrier, density as a function of temperature were also performed, using a ten-band k /spl middot/ p Hamiltonian. Together with the experimentally determined defect-related, radiative, and Auger currents we deduce the temperature variation of the respective recombination coefficients (A, B, and C). These are compared with theoretical calculations of the coefficients and good agreement is obtained. Our results suggest that by eliminating the dominant defect-related current path, the threshold current density of these GaInNAs-GaAs-based devices would be approximately halved at RT. Such devices could then have threshold current densities comparable with the best InGaAsP/InP-based lasers with the added advantages provided by the GaAs system that are important for vertical integration.     

Theoretical and experimental analysis of 1.3-/spl mu/m InGaAsN/GaAs lasers

We present a comprehensive theoretical and experimental analysis of 1.3-/spl mu/m InGaAsN/GaAs lasers. After introducing the 10-band k /spl middot/ p Hamiltonian which predicts transition energies observed experimentally, we employ it to investigate laser properties of ideal and real InGaAsN/GaAs laser devices. Our calculations show that the addition of N reduces the peak gain and differential gain at fixed carrier density, although the gain saturation value and the peak gain as a function of radiative current density are largely unchanged due to the incorporation of N. The gain characteristics are optimized by including the minimum amount of nitrogen necessary to prevent strain relaxation at the given well thickness. The measured spontaneous emission and gain characteristics of real devices are well described by the theoretical model. Our analysis shows that the threshold current is dominated by nonradiative, defect-related recombination. Elimination of these losses would enable laser characteristics comparable with the best InGaAsP/InP-based lasers with the added advantages provided by the GaAs system that are important for vertical integration.     

CPA/Tiger-MGP: test-goal set partitioning for efficient multi-goal test-suite generation

Software model checkers can be used to generate high-quality test cases from counterexamples of a reachability analysis. However, naïvely invoking a software model checker for each test goal in isolation does not scale to large programs as a repeated construction of an abstract program model is expensive. In contrast, invoking a software model checker for reaching all test goals in a single run leads to few abstraction possibilities and thus to low scalability. Therefore, our approach pursues a test-suite generation technique that incorporates configurable multi-goal set partitioning (MGP) including configurable partitioning strategies and simultaneous processing of multiple test goals in one reachability analysis. Our approach employs recent techniques from multi-property verification in order to control the computational overhead for tracking multi-goal reachability information. Our tool, called CPA/Tiger-MGP, uses predicate-abstraction-based program analysis in the model-checking framework CPA checker.

     

Modal refractive index of 1.3 μm InGaAsP, AlGaInAs and GaInNAssemiconductor lasers under high hydrostatic pressure

The accurate experimental determination of the modal refractive index of 1.3 μm InGaAsP, AlGaInAs and GaInNAs multiple quantum-well lasers by measuring the longitudinal mode separation up to a pressure of 15 kbar is reported for the first time. A small increase of the indices is observed for the three materials with increasing pressure, which causes a decrease of lasing wavelength     

Novel P-I-N-P top-emitting organic light-emitting diodes with enhanced efficiency and stability

An additional p-doping layer is added to the P-I-N stack of top-emitting organic light-emitting diodes (TEOLEDs) to control the electron tunneling current and improve interfacial stability. In addition, double p-doped layers, which are adjacent to the bottom-anode, are introduced to simultaneously optimize robustness and doping efficiency of p-type doping. In the emissive layer (EML), a second assistant emitter molecule is used which transfer its triplet energy to the actual emitter which is lower in energy, thus increasing the luminous efficacy. Such a co-doped dual-emitter layer is able to separate polarons and excitons and thus reduces chemical degradation. Compared to conventional P-I-N TEOLEDs, our novel P-I-N-P device shows negligible increase of driving voltage at low bias but offers significantly increased efficiencies. In addition, the P-I-N-P stack renders the electrical properties less sensitive to thickness variations and prolonged operation, which is attributed to the existence of a one-sided abrupt N-P tunneling junction beneath the top cathode contact.