凝血酶生成动力学检查方法实验初探

目的：探讨凝血酶生成动力学检测方法在血栓与止血研究领域的意义，在传统检查方法的基础上，通过动力学图形的标准化处理获得更为直观，丰富的实验参数。方法：利用分光光度计连接微机对弟因过程进行扫描，并绘制动力学图形，然后人工测量动力学图形自设参数。结果；研究资料显示，在一定条件下分析动力学图形中的主要分析参数与凝血酶的形成速度与量成正，负相关，并有满意的精确度[CV=(1.2%-6.6%)]与敏感性（可能最低因子含量0．325％。结论：凝血酶生成动力学检查方法可行实用，有望成为评价人体凝血功能检查方法的一种新趋势。     

Sequence polymorphisms of forensic Y-STRs revealed by a 68-plex in-house massively parallel sequencing panel

Sequence polymorphisms of Y chromosome short tandem repeat (Y-STR) markers can be unveiled using next generation sequencing (NGS). Compared to capillary electrophoresis, NGS has the advantage of distinguishing between some alleles of the same length. Here, a 68-plex in-house panel covering 67 Y-STR loci and the sex determinant Amelogenin locus, was developed. The accuracy of this panel was 100% concordant with three standard reference samples. The sensitive was as low as 250 pg. A total of 466 length-based alleles, 806 sequence-based alleles, and 149 haplotypes were observed across 149 Chinese Han individuals. The total haplotype diversity and discrimination capacity was 1.0000 in detected samples. The DYS710 locus possessed the highest diversity by sequence among these Y-STRs, with 109 sequence-based alleles observed. Micro-variant alleles with the same length were observed in 39 Y-STR loci, with their sequence variations mainly attributable to repeat pattern variations. While the number of sequence-based alleles identified for DYS447, DYS449, DYS710, DYS720 and DYF387S1a/b was approximately three times that of their length-based alleles, flanking sequence variations were observed in 18 alleles. In addition, 201 sequence-based alleles in 42 loci were newly discovered. This significantly expanded the knowledge of human Y-STR sequence polymorphisms. Collectively, the 68-plex panel provided reliable Y-STR results as well as higher resolution for paternal lineage analysis.     

Contractile activity and prostacyclin generation in isolated coronary arteries from diabetic dogs

Summary The present study was aimed at determining the generation of prostacyclin (PGI₂)-like-material in coronary arteries from normal and diabetic (pancreatectomized) dogs as well as the contractile responses to prostacyclin of preparations from normal, diabetic and insulin-treated diabetic animals. PGI₂ produced a dose-dependent relaxation of coronary arteries from normal dogs. In contrast, those from diabetic animals were not relaxed; indeed, at low concentrations PGI₂ failed to evoke any effect but at higher ones it induced a distinct contraction. In arteries from diabetic animals treated with insulin, PGI₂ induced a biphasic contractile effect, which lay between that of normal controls and untreated diabetics. In addition the basal generation of PGI₂-like-material by coronary arteries was significantly higher in the diabetic (141±0.2 pg/mg, mean±SEM) than in normal dogs (59±0.2 pg/mg). The present experiments demonstrate that the generation of PGI₂-like-substance is significantly increased in coronary arteries from diabetic dogs, but the same vessels are unable to respond to added authentic PGI₂ with relaxation; on the contrary they react with a distinct positive contractile response.     

Genetic insight into cytochrome P450 in Chinese from the Chinese Millionome Database

Genetic variations of cytochrome P450 (CYP) influence the inter‐individual differences in drug response. Here, we collected 8682 variants of 57 CYP genes and cytochrome P450 oxidoreductase (POR) from a large‐scale sequencing project in Chinese, Chinese Millionome Database (CMDB). In addition, 52 294 variants from the Genome Aggregation Database (gnomAD) had been simultaneously identified and analysed. Rare variants with a variant allele frequency (VAF) < 0.01 comprised 41.4% (3594/8682) of identified variations in the CMDB, while 98.1% (51 320/52 294) in the gnomAD were rare. Out of 8682 variants in the CMDB, 66.9% (5808/8682) were in introns and only 4.3% (377/8682) were missense variants. In contrast, 36.2% (18 929/52 294) variants in the gnomAD were missense. The common alleles with a VAF over 0.1 were found in CYP1A2*1C, CYP1A2*1F, CYP2C19*2, CYP2D6*2, CYP2D6*10, CYP3A5*3 and CYP4F2*3, with a VAF of 0.161, 0.6, 0.27, 0.274, 0.678, 0.92 and 0.233, respectively. The growing number of genetic variations in CYP genes as more genomes are sequenced would increase the power to predict drug metabolism and response based on the genotype of the particular individual.     

PCR-based next-generation West Nile virus sequencing protocols

The epidemiology of West Nile virus (WNV) is unpredictable and changing. Availability of whole genome sequences enables the detailed molecular epidemiology studies and the evaluation and design of diagnostic tools. In the present study we provide two PCR-based protocols which can be applied directly on biological samples from hosts infected by WNV strains belonging to lineage 1 or lineage 2. It was shown that the protocols worked successfully even on samples with relatively low viral load.     

Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing

BackgroundMismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision‐making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories.MethodsCRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next‐generation sequencing (NGS) and were then used to prepare formalin‐fixed, paraffin‐embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing.ResultsWe successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples.ConclusionsWe successfully established MLH1 protein‐deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.     

Exploring the utility of whole‐exome sequencing as a diagnostic tool in a child with atypical episodic muscle weakness

The advent of whole‐exome next‐generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½‐year old female patient with a 2‐year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work‐up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di‐deoxy sequencing. WES revealed a de novo non‐synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.