Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a) Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

Voltage-gated sodium channels (VGSCs; Na_V1.1–Na_V1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC₅₀ values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on Na_V1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC₅₀ value of 5.12 nM, compared with IC₅₀ values of 61.7–2700 nM for other heterologously expressed Na_V1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on Na_V1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.     

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.KEY WORDS: RNAi, Cytochrome P450, CYP3A4, shRNA, Chemosensitivity     

64例儿童门静脉高压肝脏病理学分析

目的探讨儿童门静脉高压的临床病理特征。方法对64例儿童门静脉高压的临床情况及肝组织病理形态进行分析。结果64例儿童门静脉高压患儿年龄从1岁3个月至14岁,平均年龄6.3岁。病程2 d至8年,临床主要表现为消化道出血50例(78.1%);脾肿大59例;肝肿大5例;发热8例。B超及CT检查提示门静脉海绵变性57例(89.1%);脾大59例(92.2%);8例提示肝硬化。病理学检查,57例门静脉海绵变性肝脏组织主要表现为门静脉高压的压力相关性形态学改变,其中6例合并其他肝脏病变。7例肝性门静脉高压中包含先天性肝纤维化2例,小结节性肝硬化2例,肝外胆道闭锁致胆汁性肝硬化1例,结节再生性增生1例,非硬化性门静脉高压1例。54例获得随访。结论儿童门静脉高压病因与成人不同,肝组织活检是确诊的重要手段。     

High-coverage SARS-CoV-2 genome sequences acquired by target capture sequencing

In this study, we designed a set of SARS-CoV-2 enrichment probes to increase the capacity for sequence-based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS-CoV-2 enrichment probe set contains 502 120 nt single-stranded DNA biotin-labeled probes designed based on all available SARS-CoV-2 viral sequences and it can be used to enrich for SARS-CoV-2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from cell culture, three nasopharyngeal swab samples (cycle threshold [C_t] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID-19 (positive control) and four throat swab samples from patients without COVID-19 (negative controls), respectively. Moreover, based on these high-quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication and its phylogenetic relationship with other selected high-quality samples from the Genome Variation Map. Therefore, this universal SARS-CoV-2 enrichment probe system can capture and enrich SARS-CoV-2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles.     

抗生素诱导的肠道菌群失调加重小鼠肺炎支原体感染

目的初步探讨抗生素诱导的肠道菌群失调对肺炎支原体(Mycoplasma pneumoniae,Mp)呼吸道感染的影响。方法C57BL/6J小鼠口服万古霉素和庆大霉素21 d后滴鼻感染Mp。实时荧光定量PCR(qPCR)分析抗生素处理前后小鼠粪便中5个主要菌门的菌量;qPCR检测感染后3 d和7 d肺组织中Mp载量;病理切片和HE染色分析肺组织炎症病理改变;流式细胞术分析小鼠脾脏T淋巴细胞分泌的IFN-γ和IL-4;间接ELISA检测Mp特异性IgM和IgG。结果口服万古霉素和庆大霉素后小鼠粪便中拟杆菌门菌量显著减少,厚壁菌门菌量增多,δ,γ变形杆菌门、放线菌门和软壁菌门菌量亦发生改变;口服抗生素小鼠感染Mp后3 d和7 d肺组织Mp载量和炎症病理评分增加,7 d小鼠脾淋巴细胞中分泌IFN-γ的CD4+T细胞减少。结论抗生素诱导的小鼠肠道菌群失调可加重Mp呼吸道感染。     

数据驱动的三叉神经纤维束自动分割算法

目前三叉神经的纤维跟踪成像过程中普遍存在人工依赖性问题,主要包括人工绘制感兴趣区域(ROI)及手动筛选目标纤维束,导致结果的不确定性和数据误差。针对此类问题,提出一种数据驱动的三叉神经纤维自动分割算法。利用多组大脑样本的纤维数据,建立数据驱动的纤维聚类图谱,实现新样本纤维数据的自动分割,直接得到三叉神经纤维束。在实验中,选择25组青年健康人的数据作为样本数据。首先,利用FSL软件分割工具提取脑干作为ROI,进行确定性纤维跟踪。其次,通过对20组纤维数据进行多样本配准和谱聚类,创建数据驱动的纤维聚类图谱。根据三叉神经细小的特点,在建立纤维图谱过程中,通过对脑干纤维束进行二次分类来标注三叉神经纤维束。最后,选择5组青年健康人的新样本数据,将其脑干纤维数据应用纤维图谱自动分割得到三叉神经纤维束,并计算同一样本数据的自动分割结果与手动分割结果之间的加权Dice系数。结果显示,所提出的方法成功分割5组数据的三叉神经纤维束,而传统人工方法成功识别4组三叉神经纤维束,两者结果之间的加权Dice系数分别为0.865,0.939,0.824,0.942。该方法可以有效避免人为因素的影响,提高神经外科医生与颅神经研究者的工作效率。     

一例15q11.2微重复患儿的临床及遗传学分析

目的明确1例发育迟缓、智力低下患儿遗传学病因及其来源,并对该家系下一胎行产前诊断。方法采集患儿及其父母外周血进行常规G显带核型分析及单核苷酸多态性微阵列芯片(single nucleotide polymorphism array,SNP array)检测;并对该孕妇行产前诊断,进行羊水细胞染色体核型分析及SNP array检测。结果患儿及其父母染色体核型未见异常。SNP array检测结果显示患儿15号染色体15q11.2区段存在855.3 kb重复,该重复遗传至表型正常的母亲,父亲检测结果未见异常。孕妇羊水细胞染色体核型及SNP array检测结果均未见异常。结论15q11.2微重复可能与体格/智力发育障碍相关,CYFIP1可能是其候选基因,但该重复仅可增加其发病风险,外显率较低,在临床咨询中应引起重视。     

新型3D打印导板辅助颈椎前路双侧椎弓根置钉的安全及可行性

文题释义：3D 打印手术导板：该导板依据手术需要,通过计算机辅助设计、3D打印制备出的一种具有术中准确定位点、置钉的位置、方向和深度,精确建立孔道、截面、空间距离、相互成角关系及其他复杂空间结构等功能的辅助手术器械,具有获取途径方便、使用方法简单、价格低廉等优点。 颈椎前路椎弓根置钉：是一种区别于传统颈椎后路椎弓根螺钉置钉技术的新型置钉技术,该技术可通过单独前方入路实现颈椎三柱损伤或多节段病变的坚强固定,无需再行后路固定,可有效减少手术创伤和并发症。 背景：下颈椎前路椎弓根螺钉固定技术可通过前方入路实现颈椎坚强固定,具有较大的应用前景。但该技术操作难度大、风险高,目前尚未得到广泛应用。 目的：改良设计一种用于下颈椎双侧前路椎弓根螺钉置钉的3D打印导板,探讨其辅助颈椎前路椎弓根螺钉置钉的可行性及安全性。 方法：选取6具正常成人颈椎标本,男女各3具,行薄层CT扫描后将影像数据以DICOM格式导入Mimics 17.0软件,三维重建后模拟设计出C₃-C₇双侧颈椎前路椎弓根螺钉钉道导孔,再以颈椎椎体前面、椎体上面前1/2及双侧钩突关节面前1/2骨性结构为标志,反向增厚设计生成导孔基座,形成颈椎前路椎弓根螺钉置钉导板。经3D打印得到导板实体后,在导板辅助下行C₃-C₇双侧颈椎前路椎弓根螺钉置钉。将置钉后的颈椎标本再次行CT扫描,通过CT断面影像评价置钉准确性。同时利用Mimics17.0软件比较实际钉道与模拟钉道在横断面的内、外偏移角度(α1、α2)差异及其在矢状面的上、下偏移角度(β1、β2)差异。 结果与结论：①双侧共计60枚颈椎前路椎弓根螺钉均顺利置入,其中57枚完全位于椎弓根皮质内,判定为0级,准确率95.0%;另外3枚破出椎弓根皮质,其中1级2枚(3.3%),2级1枚(1.7%);②真实钉道与模拟钉道相比,其横断面内、外偏移角分别为(0.867±0.787)°、(0.783±0.792)°,差异无显著性意义(P > 0.05);矢状面上、下偏移角分别为(1.362±1.380)°、(1.314±1.300)°,差异无显著性意义(P > 0.05)。③提示在设计得到的3D打印导板辅助下可顺利完成下颈椎双侧前路椎弓根螺钉置钉,且具有良好的置钉安全性。 ORCID: 0000-0003-0124-7585(肖强)中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程