Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.     

影像设备电路板测试与故障诊断之系统实现

大型医学影像设备是医院中的贵重设备,其中不少进口设备的维护修理的技术难度较高,医院每年花费在医学影像设备上的维护修理费是非常之大。本文从建立国内的维修力量着眼,研讨医学影像设备维修方法。     

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.