遗传病二代测序临床检测全流程规范化共识探讨(2)——样品采集处理及检测

二代测序技术(next generation sequencing,NGS)具有极高的检测通量,相对低的检测成本,高度的准确性(accuracy)和精准性(precision),是遗传病临床检测的有力工具之一。NGS实验室的检测流程是否规范,将直接影响NGS数据的稳定性、可靠性和有效性,将决定其是否能被用于遗传病的临床辅助诊断或筛查。因此.作为遗传病基因检测的重要环节之一,NGS实验室中流程的规范化与标准化非常重要。2019年5月,在第二届基因检测联盟会议上,针对如何规范NGS检测流程,从事遗传病临床诊治、实验室检测以及第三方基因检测机构的专家进行了全面充分的讨论,旨在规范基于NGS的基因检测流程,对检测流程中的前、中、后三个阶段(包括样本采集/接收/保存、NGS建库、上机测序及数据质控)的操作与实施提出了专业性的指导意见,以规范NGS技术在遗传病基因检测领域中的应用。本文根据此次研讨会上各行业专家的讨论,总结并发布NGS实验室检测流程的规范共识,以促进NGS实验室流程的规范化和标准化,推进我国NGS实验室在遗传病基因检测领域的快速和专业化发展。     

Automatic modeling of pectus excavatum corrective prosthesis using artificial neural networks

Pectus excavatum is the most common deformity of the thorax. Pre-operative diagnosis usually includes Computed Tomography (CT) to successfully employ a thoracic prosthesis for anterior chest wall remodeling. Aiming at the elimination of radiation exposure, this paper presents a novel methodology for the replacement of CT by a 3D laser scanner (radiation-free) for prosthesis modeling.The complete elimination of CT is based on an accurate determination of ribs position and prosthesis placement region through skin surface points. The developed solution resorts to a normalized and combined outcome of an artificial neural network (ANN) set. Each ANN model was trained with data vectors from 165 male patients and using soft tissue thicknesses (STT) comprising information from the skin and rib cage (automatically determined by image processing algorithms). Tests revealed that ribs position for prosthesis placement and modeling can be estimated with an average error of 5.0 ± 3.6 mm. One also showed that the ANN performance can be improved by introducing a manually determined initial STT value in the ANN normalization procedure (average error of 2.82 ± 0.76 mm). Such error range is well below current prosthesis manual modeling (approximately 11 mm), which can provide a valuable and radiation-free procedure for prosthesis personalization.     

Bioactive nanoparticle embedded microcapsules for improving the efficacy of type I diabetes therapy

Abstract

In order to overcome the side effects of pancreatic transplantation and insulin injection treatment for type I diabetes, we established a drug delivery system employing nanoparticle embedded microcapsules (NEMs). The system co-encapsulated chitosan nanoparticles with γ-aminobutyric acid and β-TC-6 cells for combined drug and cell therapy in diabetes mellitus (DM). The NEMs, which were formed via high-voltage electrostatic method, had an excellent sphericity with a smooth surface. The average size NEM was 245.52?±?22.00?μm, which indicated a good size for cell encapsulation. Haemolysis rate of NEMs at concentrations of 100, 200 or 300?mg/mL were all below 5%. Relative viability rates of L929 cells with the same concentrations at 24, 48 or 72?h were all above 80%. We implanted bioactive NEMs into type 1?DM mice to evaluate the effect of the combined therapy. The level of blood glucose in the group receiving the combined therapy decreased during the first 2?weeks of treatment. During the next week, the level of blood glucose stayed in a safe range. Body weight continuously increased during the postoperative period after combined therapy group. Oral glucose tolerance test (OGTT) performed after 24?d showed that the level of blood glucose combined therapy reached the maximum peak of 13.04?mmol/L, lower than 16.56?mmol/L for the cell therapy group. This primary study indicated that microencapsulation technology and combined therapy are promising for the treatment of type I diabetes mellitus.     

Analysis of oocyte cryopreservation in assisted reproduction: the Italian National Register data from 2005 to 2007

This paper reports on oocyte cryopreservation efficacy in Italy with respect to successful IVF from 2005 to 2007, presenting data from 193 centres collected by the Italian National Register. Post-thawing survival rates, number of transferred embryos, implantation rates and clinical pregnancy rates per transfer with respect to frozen/vitrified oocytes (FVO) were analysed. These numbers were compared with those obtained using frozen embryos or fresh oocytes. A total of 121,708 cycles were initiated, of which, 7.1% (8682) were FVO cycles and 2.4% (2952) were frozen embryo cycles. Of the 81,786 FVO, 52.5% (42,917) were thawed and 26.9% (22,005) inseminated. Of those inseminated, 68.0% (14,966) yielded good embryos. These numbers were significantly lower than those using fresh oocytes in which 77.9% (197,242; fresh oocytes versus FVO P < 0.001) of inseminated oocytes generated good embryos. Implantation rate using FVO was 6.9%, which was significantly lower than that using fresh oocytes (13.5%; P < 0.001) and frozen embryos (8.8%; P < 0.001). Pregnancy rate per transfer using FVO was 12.5% and significantly lower than that using fresh oocytes (24.9%; P < 0.001) or frozen embryos (16.4%; P < 0.001). There were 505 deliveries after IVF with FVO and 582 babies.     

肺腺癌实性结节肿瘤间质与多排螺旋CT早期增强间的关系研究

Objective: To evaluate correlations between proportion and distribution of tumor stroma and MDCT early phase enhancement character in solid lung adenocarcinoma, and compare with microvessel density and histological subtypes.Methods: Thirty-one patients with lung adenocarcinoma shown as solid solitary pulmonary nodules underwent routine contrast-enhanced MDCT followed by surgical resections. CT character included net enhancement and distribution of enhancement. The largest cut surface of tumor specimens were stained by hematoxylin and eosin. About 25 fields of view of each specimen were scanned as digitized pictures at low magnification. Semi-auto segmentation software was used to calculate mean stroma proportion. Pearson correlation coefficient was used to represent the relationships between extent of tumor enhancement, proportion of tumor stroma and MVD respectively. Fisher's exact test was used to analyze statistical differences in degree of CT enhancement among groups of different histological subtypes. Results: Proportion of invasive tumor stroma (13.2%-54.5%, mean 26.2 ± 8.8%) was correlated positively with net enhancement (8-60.8 HU, mean 31.2 ± 13.6 HU; r =0.483, P= 0.006) which was more than MVD. 58.1% cases showed homogenous enhancement, 32.3% cases showed peripheral inhomogenous enhancement, 3.2% cases showed central inhomogenous enhancement, 3.2% cases showed asymmetrical inhomogenous enhancement, and 3.2% cases showed no enhancement. 58.1% cases' stroma showed mixed distribution,35.5% cases showed peripheral distribution, 3.2% cases showed central distribution, and 3.2% cases showed asymmetrical distribution. Significantly more adenocarcinomas classified with "net enhancement ＞ 20 HU" were found in the acinar group than in the solid with mucin subtype (P = 0.005). Conclusion: Extent of CT enhancement reflects underlying not only the tumor angiogenesis but also stroma proliferation in solid small lung adenocarcinoma. Tumor stroma proportion could reflect the histopathologic basis of small lung adenocarcinoma's CT enhancement substantially than MVD. Distribution between enhancement and tumor stroma have good correspondence. Most of acinar adenocarcinomas have higher degree of CT net enhancement than solid with mucin adenocarcinomas.     

吉西他滨联合铂类治疗64例晚期非小细胞肺癌临床疗效研究

目的:比较吉西他滨(gemcitabine)联合顺铂(cisplatin)、卡铂(carboplatin)和奥沙利铂(oxaliplatin)三种化疗方案对晚期非小细胞肺癌(NSCLC)的疗效和毒性反应。方法:经病理和细胞学证实的64例晚期NSCLC患者随机分为吉西他滨 顺铂(gemcitabine cisplatin,Gcis)、吉西他滨 卡铂(gemcitabine carbopl-atin,Gcarb)和吉西他滨 奥沙利铂(gemcitabine oxaliplatin,GLOHP)三组。三组均选用吉西他滨1000mg/m2静脉滴注第1、8天。GCis组:顺铂70mg/m2静脉滴注,第1天;GCarb组:卡铂AUC4～6(初治6,复治4～5),静脉滴注,第1天;GLOHP组:奥沙利铂LOHP130mg/m2静脉滴注,第1天。三组均21天为一周期,连续使用2～3周期评价疗效和毒副反应。结果:Gcis、Gcarb、GLOHP三种方案治疗晚期非小细胞肺癌的有效率分别为52.38%(11/21)、50.00%(10/20)和60.87%(14/23)(P>0.05)。三种方案毒副反应主要为可耐受的骨髓抑制、消化道反应、脱发和外周神经毒性等。结论:吉西他滨联合三种不同铂类的化疗方案均为治疗晚期非小细胞肺癌较为安全有效的化疗方案。     

姜黄素对II相酶GST及NQO酶活性的诱导及其机制

研究姜黄素对II相酶谷胱甘肽转移酶(GST)及NADP(H)醌氧化还原酶(NQO)活性的影响及其诱导机制。用光谱法检测细胞GST酶和NQO酶的活性，以及还原型谷胱甘肽(GSH)的含量；利用蛋白印迹法检测核转录因子Nrf2在胞浆与胞核的分布；采用凝胶电泳迁移率分析法(EMSA)检测Nrf2与II相酶基因抗氧化反应序列(ARE)结合活性。不同浓度的姜黄素(10～30 μmol·L^-1)刺激结肠腺癌HT-29细胞后，能显著诱导GST酶及NQO酶活性的增加，同时能迅速提高细胞内GSH的含量；蛋白印迹和凝胶电泳迁移率结果显示，姜黄素诱导细胞核内转录因子Nrf2积聚，Nrf2-ARE的结合活性增加。姜黄素诱导的II相酶GST酶及NQO酶活性增加与促进转录因子Nrf2由胞浆向胞核发生转位分布和增强Nrf2-ARE结合活性有关。     

Quantitative determination of zidovudine diaryl phosphate triester pro-drugs in rat plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A rapid, simple, and sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS) method was developed and validated for quantitative analysis of 3′-azido-3′-deoxythymidine (zidovudine, AZT) diaryl phosphate triester pro-drugs, in rat plasma using 2′,3′-dideoxy-2′3′-didehydrothymidine (d4T) as internal standard (IS). The analytes were extracted from rat plasma with methanol after protein precipitation. The compounds were separated by HPLC with gradient elution (on a Shim-pack VP-ODS C₁₈ analytical column using a mobile phase of methanol/10 mM ammonium acetate). All the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQs were 10 ng mL⁻¹ for M1, M2, M3, M4, and M5, respectively. Correlation coefficient (r) values for the linear range of 10–10,000 ng mL⁻¹ were greater than 0.999 for all the analytes. The intra-day and inter-day precision and accuracy were higher than 7.13%. The relative and absolute recovery was above 72% and no matrix effects were observed for all the analytes. This validated method provides a modern, rapid, and robust procedure for the pharmacokinetic studies of the pro-drugs after intravenous administration to rats. Some important results of AZT diaryl phosphate triester pro-drugs concerning chemical effect on pharmacokinetic performance are also studied.