Mass-Dependent Critical Value Expressions for Particle Finding in Single-Particle ICP-TOFMS.

In time-of-flight mass spectrometry (TOFMS), ion detection is often achieved via electron multiplication followed by fast analog-to-digital conversion (ADC). This detection approach is chosen over time-to-digital conversion because it extends the dynamic range of TOFMS measurements, especially for transient analyses. However, fast ADC detection also introduces measurement noise fundamental to the electron multiplication process. In previous research, we demonstrated that TOFMS signals acquired with fast ADC follow a compound Poisson distribution in which the Poisson-distributed arrival of ions at the detector is compounded with the response profile of the electron multiplier. Here, we consider the influence of mass-to-charge (m/z)-dependent detector responses and their impact on particle-finding accuracy in single-particle inductively coupled plasma TOFMS (spICP-TOFMS). In spICP-TOFMS, highly time-resolved ion signals are recorded and particle signals are distinguished from background signals based on thresholding the data at m/z-specific critical values. Through Monte Carlo modeling with measured m/z-dependent detector responses, we generate compound Poisson model distributions and critical values that accurately account for the dispersion of measured signals. We test the accuracy of critical values through the analysis of dissolved element solutions and comparison of measured versus predicted event rates above critical value thresholds. The use of m/z-dependent compound Poisson critical values reduces false-positive particle identifications by one to two orders of magnitude compared to thresholding criteria based on normal or Poisson statistics. The improved accuracy and robustness of compound Poisson critical values enables automated multi-element particle finding in spICP-TOFMS.

Comparison of segmental fracture healing in young and old rats that were treated with bone stimulators - biomed 2009.

Demineralized bone matrix (DBM) has been shown to have osteoinductive properties, and demonstrated accelerated rate of bone regeneration. However, prior studies using DBM in young rats have not been consistent with results witnessed in an older rat population. There were two aims of this study: (1) to compare segmental bone defect healing in old rats treated with DBM versus young rats treated with DBM over time, and (2) to compare the number of cell types present at the fracture site in young and old animals during fracture repair. DBM was delivered over time with an initial burst of substances and subsequent sustained release over time using the calcined tricalcium phosphate lysine (TCPL) capsules. In addition, the capsules contained antibiotics to prevent infection. The fracture sites were evaluated for evidence of fracture healing by radiographs and histomorphometric techniques after 4, 8, and 15 weeks. Animals treated with DBM had smaller fracture callus and mature bone present with similar number of osteoblast as control at 15 weeks; whereas animals treated with antibiotic alone had limited bone formation with time. Further studies are needed to investigate the mechanisms leading to bone formation in the presence of growth promoting factors.

Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay-accelerating factor.

BACKGROUND: The expression of human complement regulators has been proved as an effective strategy to overcome hyperacute rejection in discordant xenogeneic organ transplantation. In this study, we tested the hypotheses that expression of triple transgenes for human complement regulators and provide more effective protection to the transplanted pig tissues. METHODS: Pigs transgenic for human complement regulatory proteins, human CD59 (hCD59) and human membrane cofactor protein (hMCP), have been generated using large genomic constructs. Heterozygous human decay-accelerating factor (hDAF) transgenic pigs, from a previously established line, were bred with hCD59 or hCD59 plus hMCP pigs to produce animals that expressed both hCD59 and hDAF, or expressed triple transgenes hCD59, hDAF and hMCP. RESULTS: All three transgenes were widely expressed in most of the tissues analyzed, but the expression of hMCP was at low levels. In cytotoxicity assays on porcine peripheral blood mononuclear cells, the expression of a single transgenic protein, hCD59, or hCD59 in combination with hMCP provided similar protection against human complement-mediated damage as the single expression of hDAF. However, the expression of triple transgenic proteins or double hCD59 and hDAF transgenic proteins provided greater protection than either hCD59 or hDAF alone. CONCLUSIONS: Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.

Production and characterization of a pig line transgenic for human membrane cofactor protein.

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.