β1肾上腺素能受体反义寡核苷酸对自发性高血压大鼠心肌间质及左室功能的影响

目的观察在自发性高血压大鼠中以阳离子脂质体载体介导的β1肾上腺素能受体反义寡核苷酸逆转其心肌纤维化及改善其左室舒缩功能的作用。方法20只雄性19周龄SHR随机分为阳性对照组(n=6),反义(AS-ODN)治疗组(n=7,尾静脉β1-AS-ODN1.0mg/kg20天内共3次),反向(IN-ODN)治疗组(n=7,尾静脉注射β1-IN-ODN1.0mg/kg20天内共3次),另选同龄雄性WistarKyoto(WKY)大鼠为正常对照(n=6)。常规方法测定尾动脉收缩压、左室血流动力学指标及左心室重量指数(LVMI),显微荧光技术观察FITC标记的寡核苷酸在心肌细胞内的摄取及分布,VanGieson染色法检测左室胶原容积分数(CVF),RT-PCR法检测左室心肌β1肾上腺素能受体(β1-AR)mRNA表达。结果(1)转染后3d,寡核苷酸被心肌细胞有效摄取,均匀分布于其胞浆内;(2)与WKY组大鼠相比较,28周龄SHR存在高血压,心肌肥大、纤维化及左室功能不全;(3)与SHR组比较,β1-AS-ODN治疗组大鼠左室心肌β1肾上腺素能受体(β1-AR)mRNA表达减少,血压及LVMI、CVF降低,左室舒缩功能得到改善。结论β1-AS-ODN通过在转录水平抑制SHRβ1-AR的表达,在持久而有效降压的同时,更能有效地逆转左室肥大、纤维化及改善左室功能。     

2008－2017年鄂西北地区脑膜炎奈瑟菌药物敏感性及分子分型研究

目的 分析鄂西北地区2008 – 2017年分离到的72株脑膜炎奈瑟菌（Nm）的药物敏感性及分子分型特征。 方法 对Nm菌株进行生化鉴定和血清学分群,并采用荧光聚合酶链式反应进行基因分群,用K-B纸片和E-test检测试纸条进行药敏试验;选取部分菌株进行多位点序列分型（MLST）和脉冲场凝胶电泳（PFGE）。 结果 血清分群结果为B群14株、C群10株、W群3株、E群1株、不可分群44株。基因分群结果:B群33株、C群10株、W群3株、E群1株,其他及不可分群25株。70株Nm菌株对复方新诺明、萘啶酸、环丙沙星、米诺环素、氯霉素5种抗生素的敏感率分别为7.14%、21.43%、28.57%、98.57%和98.57%,对美罗培南、亚胺培南、阿奇霉素、头孢曲松、头孢噻肟、青霉素和利福平均敏感。17株菌株共有12种序列型别,其中12株菌株不能归入现有克隆群,其他5株属于CC4821（4株）和CC175（1株）。55株菌株分为48种不同的PFGE带型,带型相同的菌株其基因群亦相同。 结论 2008 – 2017年鄂西北地区分离的Nm菌株主要为B群;对复方新诺明、萘啶酸和环丙沙星具有较高的耐药性。MLST分型以CC4821为优势克隆。PFGE分型特征呈多态性,未出现优势带型。      

加德纳菌与临床细菌性阴道病的相关性

目的研究阴道加德纳细菌(GV)与细菌性阴道病(BV)的关系,探讨GV对BV诊断的病原学价值及GV感染的相关危险因素。方法以489例BV患者为研究对象(BV组),217例健康体检妇女为对照组,观察两组白带线索细胞阳性率、GV荧光抗体检测阳性率及BV试验,研究BV相关高危因素。结果 BV组线索细胞阳性率(94.1%)、GV荧光抗体检测阳性率(87.3%)、BV试验阳性率(74.8%)、pH值(4.5±0.3)与对照组差异显著(均P<0.01);logstic多因素回归分析显示,流产史、年龄>40岁为GV感染的独立高危因素;文化程度为GV感染的保护性因子,文化程度越高,GV感染机率越小。结论 GV与BV的发生高度相关,流产史、年龄>40岁为GV感染的独立高危因素。     

高效液相色谱法测定克拉维丁搽剂中醋酸地塞米松含量

目的测定克拉维丁搽剂中醋酸地塞米松的含量。方法用高效液相色谱法,色谱柱为C18柱(150 mm×4.6mm,5μm);流动相为甲醇-水-冰醋酸(80200.5);检测波长为240nm;进样量20μl;柱温30℃;流速1ml/min。结果醋酸地塞米松在2.00~64.00μg/ml范围内线性关系良好,Y=0.405 2 X+0.804 2(r=0.999 9),平均回收率为99.3%,RSD=0.93%(n=9)。结论该法便捷,重现性好,适用于克拉维丁搽剂中醋酸地塞米松的含量测定。     

天然高分子白及溶胶的制备及质量控制

目的制备高分子白及溶胶,制定质量控制方法。方法用水提醇沉法提取,0.1%活性炭脱色,所得多糖干燥、粉碎、加纯化水制备成溶胶;从性状、鉴别、含量测定方面控制质量。结果所制得白及溶胶为浅棕色半流动状,质地均匀,细腻,具有粘性,鉴别呈正反应,溶胶中含白及多糖为4.0%(±5),含量测定多糖在1.88~33.75μg.m L-1范围内线性关系良好,y=0.034X-0.0069(r=0.9998),回收率为98.6%,RSD=1.12%(n=9)。结论该方法制备的白及溶胶均匀、细腻,质量可控。     

HPLC-DAD法测定“武当三号金银花”不同部位中芦丁和木犀草苷的含量

目的：建立HPLC－DAD法测定“武当三号金银花”不同部位中芦丁和木犀草苷的含量。方法采用Fortis Xi Phenyl柱（250 mm ×4．6 mm,5μm）;流动相为乙腈（A）—0．5％冰醋酸溶液（B）进行线性梯度洗脱;检测波长为354 nm;柱温：30℃。结果芦丁和木犀草苷在各自测定的范围内均呈良好的线性关系（r≥0．9994）,平均回收率分别为99．4％、99．3％。结论该法操作简单,灵敏度高,重现性好,为控制“武当三号金银花”不同部位芦丁和木犀草苷的质量提供了一种可靠的方法。     

BARF1表达下调通过活化caspase依赖的线粒体通路诱导EBV阳性胃癌细胞凋亡

目的:研究BARF1表达下调对EBV阳性胃癌细胞凋亡的影响,以及BARF1基因沉默介导细胞凋亡的分子机制。方法:siRNA和NCsiRNA分别转染NUGC3和SNU719细胞,运用Western blot测定细胞中BARF1、Bcl-2、Bax、细胞色素C、caspase 3和caspase 9的蛋白表达;RT-PCR测定BARF1、Bcl-2和Bax mRNA的表达;台盼蓝染色法测定细胞存活率;Annexin V-FITC/PI染色法和流式细胞仪测定细胞凋亡;细胞凋亡因子抗体芯片分析细胞中凋亡相关蛋白的表达;线粒体膜电位检测试剂盒测定线粒体膜电位;免疫共沉淀检测细胞中Apaf-1和caspase 9的相互作用。结果:与空白对照组和阴性对照组相比,BARF1基因沉默显著诱导NUGC3和SNU719细胞凋亡,而线粒体膜电位显著降低。BARF1沉默基因能促进促凋亡蛋白的表达并抑制抗凋亡蛋白的表达,Bcl-2/Bax比例显著降低;而caspase抑制剂能抑制由BARF1基因沉默介导的细胞凋亡。在siRNA转染的细胞中,caspase 3和caspase 9蛋白发生裂解,细胞色素C的浓度显著高于阴性对照组,Apaf-1蛋白与caspase 9蛋白在细胞质中能够发生相互作用。结论:BARF1基因沉默通过线粒体途径调节Bcl-2和Bax蛋白的表达进而诱导NUGC3和SNU719细胞凋亡,并呈caspase通路依赖关系。     

Tosight视频喉镜引导经口气管插管换经鼻气管插管用于重症监护中的效果

目的观察分析ICU采用Tosight视频喉镜引导下经口气管插管更换经鼻气管插管导管的方法及效果。方法选取30例ICU治疗中行经口气管插管患者为研究对象,数字法随机将患者分为两组,其中实验组15例给予Tosight视频喉镜引导下更换经口气管插管为经鼻气管插管,对照组给予传统Macintosh直接喉镜更换经口气管插管为经鼻气管插管,比较两组患者临床资料、操作及留置时间、气管插管前后血流动力学指标及并发症发生情况。结果实验组操作时间短于对照组操作时间,差异有显著性(P<0.05),留置时间略长于对照组,但与对照组比较差异无显著性(P>0.05)。对照组患者在气管插管时及气管插管后2分钟心率、舒张压及收缩压高于观察组(P<0.05),其他时间点两组各指标差异无显著性(P>0.05)。两组比较气管插管时及插管后并发症发生率差异有显著性(χ2=3.333,P<0.05)。结论 ICU采用Tosight视频喉镜引导下经口气管插管更换为经鼻气管插管与传统Macintosh直接喉镜比较暴露良好,插管操作刺激小,一次成功率较高,并发症较少,值得临床应用。     

纤维蛋白胶运用于翼状胬肉手术的临床观察

目的:观察纤维蛋白胶运用于翼状胬肉切除联合自体结膜移植术的临床效果。方法:选取60例60眼原发性鼻侧翼状胬肉患者随机分为试验组(纤维蛋白胶组)和对照组(缝线组)各30例30眼。行翼状胬肉切除联合自体结膜移植术,试验组采用纤维蛋白胶粘合固定植片,对照组采用10-0尼龙线缝合固定植片。术后随访6mo,观察手术时间、术后疼痛、异物感、并发症以及复发。结果:试验组手术时间(24.5±6.5min)较对照组(35.2±5.4min)短,两组差异有统计学意义(P<0.05)。术后患者疼痛和异物感试验组较对照组减轻(P<0.05)。两组均未发生术后严重并发症,试验组结膜下出血发生率低于控制组(P<0.05),6mo时纤维蛋白胶组1例(3%)复发,缝线组3例(10%)复发。结论:纤维蛋白胶运用于翼状胬肉手术固定结膜植片能减轻患者术后不适,减少手术时间及术后并发症,是一种安全有效的方法。     

Endonuclease IV recognizes single base mismatch on the eighth base 3′ to the abasic site in DNA strands for ultra-selective and sensitive mutant-type DNA detection

Since single nucleotide polymorphism (SNP) is related with many diseases and drug metabolic polymorphous and SNP genotyping is rising rapidly in many biological and medical areas, various methods of discriminating SNPs have been developed, one of which is an enzyme-based method. We uncovered a unique property of endonuclease IV due to which it can discriminate single base mismatches in different positions of DNA strands containing an abasic site, and we also discovered a new property: a mismatch in the +8 position could inhibit the cleavage of endonuclease IV. Then, we coupled +8 mismatch with other mismatches along with the discrimination effect of melting temperature to develop a new ultra-selective and sensitive genotyping system, which showed high discrimination factors. The detection limit was as low as 0.05–0.01%. Our new discovery improves the understanding of endonuclease IV. Also, the method could be applied to clinical real samples; thus, it merits further investigation and improvement for application in clinical utilization for early screening of specific diseases.

Endonuclease IV discriminates single basic mismatch in +8 position towards abasic site, which enables the detection of mutations in abundance of 0.01%.

Single nucleotide polymorphism (SNP) genotyping is playing a vital role in genome mapping, pharmacogenetic studies, and drug discovery.¹ One of the greatest important applications of SNP in biomedical research lies in comparing genome regions between cohorts, for example, matched cohorts with and without a disease, in genome-wide association studies. Statistics show that there exist some similar gene mutations in the diseased population of certain diseases, which are generally single gene mutations in multiple discontinuities. The detection of statistically significant single gene mutations is an important means of early screening of diseases, especially cancers.However, in some cases, such as tissue biopsy and liquid biopsy, detection may encounter obstacles because of low abundance. For instance, tumor DNA is often covered by a large amount of normal DNA; thus, it is difficult to detect. A considerable amount of research has been done to develop genotyping systems during the last decades, providing us with powerful new insights into mutation detection.Fluorescent probes are applied in genotyping and have become widely used tools; for example, molecular beacon (MB),^2–4 branch-migration-based probe,^5,6 cell penetrating peptide (CPP)–DNA fluorescent probe⁷ and triple-stem probe.⁸ Enzymatic tools have also been developed such as nicking endonuclease,⁹ DNase I,^10,11 λ exonuclease^12,13 and DNA ligases.¹⁴ Endonuclease IV (Endo IV) is one of the enzymes used for genotyping.^12,15–20 Endo IV recognizes apurinic/apyrimidinic (AP) sites and is eliminated in base excision repair (BER).^21,22 It prefers double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) while cleaving the phosphodiester bond.^23,24The excellent property of Endo IV has attracted many researchers. In 2013, Xiao et al. uncovered a novel property of Endo IV due to which it can discriminate mismatches next to the AP site in DNA strands;²⁵ they found that Endo IV rapidly cleaved dsDNA containing a mismatch 3′ to the AP site (3′ mismatch) or a mismatch 5′ to the AP site (5′ mismatch), whereas it hardly cleaved dsDNA containing both the mismatches, i.e., 3′ and 5′ mismatches. Through simple design, they synthesized a fluorophore- and quencher-labeled DNA probe containing an AP site that had 3′ mismatch to mutant-type (MT) DNA and 3′ and 5′ mismatches to wild-type (WT) DNA for the detection of single base mutation DNA with a detection limit of 0.01%.¹⁷ Without redundant components, sophisticated design and complex procedures, the method offers us an excellent biosensing platform with a relatively low limit of detection (LOD), which cannot be reached by most other assays. To simplify the narration, we define the mismatch at the mutant base as functional mismatch and the mismatch that probe-WT and probe-MT duplex both have as intrinsic mismatch; for the biosensing system mentioned above, 3′ mismatch is intrinsic mismatch, and 5′ mismatch is functional mismatch.Unfortunately, the study has a defect: when a sample shows positive result, it cannot assure that the slow cleavage has resulted from the mismatch at the position next to and 5′ to the AP site, that means, it cannot rule out the possible interference caused by a mismatch at another position in the DNA strand. Notably, for a hotspot of mutation, several adjacent bases are relatively high in mutation rate. A point mutation near the base of target mutation may be found in a sample gained from the patient; thereby, a mismatch other than target mismatch may be led in. Herein, we have discussed the following points under separate conditions: (a) if the unexpected mutant base lies in the intrinsic mismatch base, leaving the hybridization with a functional mismatch (5′ mismatch), a rise in fluorescence can be found, which produces a false positive result; (b) if the unexpected mutant base lies in other bases, three mismatches are formed by the probe and MT DNA, making the results unpredictable. The deficiency reduces the validity of clinical trials, which may be developed using this assay in the future.For condition (a), the reason for the predicament is that the inhibitory effect occurs if and only if two mismatches occur collectively, which is a double regulation; the reason is as follows: consecutive three mismatches that consist of an AP site and its two adjacent mismatches together result in a locally non-hybrid single-stranded status at the AP site, which can hardly be cleaved by Endo IV, as is mentioned above.²³ Thus, the enzymatic activity is inhibited. However, our current understanding of Endo IV limits further expansion of its usage.To solve this problem, we must find a mechanism such that the inhibitory effect of the enzyme is only regulated by a single mismatch. We speculate that some single-base mismatches at bases other than those next to the AP site may inhibit enzymatic cleavage although single-base mismatch next to the AP site cannot produce inhibitory effects, which has never been studied before.Therefore, we investigated the effect of single mismatch in every base in an AP-site-containing DNA duplex. We synthesized a 21-nt fluorophore- and quencher-labeled probe with an AP site, which is the same as Xiao et al.’s design,²⁵ and tested perfect matched and mismatched DNA strand (see Fig. 1a). Endo IV recognized AP-site-containing DNA strands and cleaved them at diverse rates, and the two resultant single strands detached from the target strand due to thermal instability, thus emitting fluorescent signals. Based on the predicted melting temperatures of the probe and two resultant strands, we set the experimental temperature at 42 °C; at this temperature, the melting temperature (see Table S1^†) of the probe-target duplex was not reached due to which they were tightly bound. The discrimination ability of fluorescent probes originated from the thermodynamic difference of single-base mismatches, but the thermodynamic difference caused by different types of base mismatches varied. We term the mismatches that lead to small thermodynamic difference as “stable single-mismatch”, and these yield small discrimination factors.^26,27 Stable mismatches are reported to be more difficult to detect; thus, we chose unstable mismatch: for base G, we studied G:A mismatch and for base C, A, and T, we selected C:C, A:C, and T:C mismatches, respectively.Open in a separate windowFig. 1(a) Schematic illustration of the effects of different mismatches in different positions of AP-site-containing DNA strands on the cleavage rate of Endo IV. The blank frame represents the AP site in fluorophore- and quencher-labeled oligonucleotide. (b) The bar chart of DNA strands with single mismatch in different positions. 0 denotes PM target strand.We surveyed mismatches in every position in the DNA strands containing an AP site, and the results showed that mismatches at different positions to the AP site had distinct influence on the cleavage rate of Endo IV. We denoted +x as the x^th nucleotide 3′ to the AP site and −x as the x^th nucleotide 5′ to the AP site. A −3 A:C mismatch slightly accelerated the reaction similar to +1 A:C mismatch. We observed that −9 T:C, −8 A:C, −5 T:C, −4 G:A, −2 A:C, −1 C:C, +3 A:C, +4 T:C, +5 G:A, +6 C:C, +9 C:C, +10 T:C and +11 T:C mismatches had negligible deaccelerating effect on the cleaving rate. All of the above-mentioned mismatches exhibited no significant difference compared to perfect match; thus, we call them normal mismatches. We observed that −7 T:C, −6 C:C, +2 G:A, +7 A:C, and +8 C:C mismatches clearly decelerated the reaction process; thus, we termed them as slow mismatches. The discrimination factor (DF) can be defined as the cleaving rate ratio of perfect match (PM) target to mismatch (MM) target; the influence on DF led by mismatch position is shown in Fig. 1b.Remarkably, the cleavage rate of +8 mismatch was extremely slow with DF of 39.5, which was feasible to perform genotyping. A novel method can be roughly developed when we regard +8 C:C mismatched DNA strand as a WT-probe duplex and PM DNA strand as a MT-probe duplex.The DF value was still not impressive enough. Then, we designed DNA strands with double mismatches consisting of +8 mismatch and a normal mismatch, and we experimented with their cleaving rates. More than expected, we found that the inhibition effect of +8 mismatch was even enhanced by adding a new normal mismatch; thus, we could expect an even larger discrimination effect. Then, we paired the normal mismatch and corresponding double mismatch (add +8 mismatch) as they had only 1 bp difference and raised the temperature between the melting temperature (T_m) of single mismatch strands and double mismatch strands to compare the cleaving rates (see Table S2^†). In this case, the resultant DFs were the co-effect of the property of Endo IV and the differentiation of thermodynamics. It should be noted that the conception of DF of a double mismatch target needs a minor modification, which defines the quotient of the cleaving rate of the corresponding single mismatch and that of double mismatches. The results are shown in Table S3.^† An exceptional discrimination was acquired between the pairs +1 A:C mismatch and +1 A:C with +8 C:C mismatch [denoted +1(A:C)/+8(C:C) mismatch hereinafter, DF = 62.30], −2 A:C and −2(A:C)/+8(C:C) (DF = 61.86), −1 C:C and −1(C:C)/+8(C:C) (DF = 127.04), and +3 A:C and +3(A:C)/+8(C:C) (DF = 877.07).We then investigated other types of base mismatches at the same positions and calculated their DFs (see Table S3^†). We concluded that for all types of +3 with +8 mismatches, DFs were remarkable, especially for +3 A:C with all three types of +8 mismatches, and their DFs ranged from 258.28 to 877.07; hence, they had the greatest potential for genotyping among all double mismatches (see Fig. 2a and b). From the results, we could deduce that DFs were indeed mainly determined by the mismatch position of a nucleotide. Interestingly, the hybridization stability of mismatch types determined DFs. It is known that for A:V (V = A, C, G) mismatch, A:G > A:A > A:C and for C:H (H = A, C, T) mismatch, C:T > C:A > C:C.^28–30 Our results (Fig. 2b) showed that DF increased with the hybridization stability; thus, we inferred that the recognition ability of Endo IV was partially derived from the thermodynamic property of base pairs. For different normal mismatches with all types of +8 mismatch, DFs were usually C:C > C:A > C:T.Open in a separate windowFig. 2(a) Schematic illustration of examining detection limit of DNA strands with double mismatches [+3(A:C)/+8(C:C) mismatch] in a large background of DNA strands with single mismatch [+8(C:C) mismatch]. (b) All types of +3(A:C)/+8 mismatches have significant inhibition effect on the cleaving rate of Endo IV, especially +3(A:C)/+8(C:C) mismatch. (c) Detection limit of DNA strands with double mismatches [+3(A:C)/+8(C:C) mismatch] in a large background of DNA strands with single mismatch [+8(C:C) mismatch]. The detection limit is 0.01%.Based on our previous study, to investigate the detection limits of the pair selected, we mixed up each double mismatch with its unique corresponding single mismatch to simulate low abundance mutant-type DNA sample, in which single mismatch represented MT target, and double mismatches represented the WT target (see Fig. 2a and c). The detection of 0.01% MT was available for +3(A:C)/+8(C:C) and +3(A:C) pair. For different directions of mutation, we could detect MT target in abundance as low as 0.05–0.01% in 42 min (see Fig. 2c, S1 and S2^†). We also examined the effect after applying it to PCR product treated with exonuclease I and λ exonuclease, and DF was the same as mentioned below (see Fig. S3^†). Three hours were required for the whole assay. We also studied the cleavage effect of Endo IV while encountering three mismatched strands containing at least one slow mismatch. The rate of cleavage was even slower than that for single slow mismatch; thus, the mismatch at an untargeted position does not have any impact on the result of the trial while using this method.We have fully proven the application of the synthesized DNA and then, we demonstrated the applicability of the method to real clinical samples. We extracted genome DNA of a colorectal adenocarcinoma sample, which was detected by BRAF gene exon 15 V600G mutation as MT DNA; then, it was diluted by wild-type genomic DNAs extracted from a normal tissue without that mutation to prepare a series of mixed samples with MT DNA at different abundances. After PCR amplification and two-step enzymatic treatments mentioned below, single-stranded DNA was produced. Then, a customized fluorescent probe was added to the solution, which formed double mismatched duplex with WT DNA and single mismatched duplex with MT DNA (see Fig. 3a). After adding Endo IV, as shown in Fig. 3b, the detection limit was 0.01%. Thus, the method is feasible for a sample gained from biopsy. The data firmly proved the applicability of the method to real clinical samples.Open in a separate windowFig. 3(a) Real clinical sample detection needs four steps: genome DNA extraction, PCR amplification, single-stranded DNA forming and Endo IV detection. (b) The detection limits of the real samples are the same as those of the synthesized DNA strand. Since it was a somatic mutation in BRAF gene, the data of sample in 100% abundance could not be collected.The new properties we discovered enriched our understanding of Endo IV. For AP site binding, Endo IV forms an eight-stranded α-β barrel fold (TIM barrel), and the active site includes three metal ions near the center of the barrel.³¹ Our current understanding is that only the bases near the center of the barrel, i.e., adjacent to the AP site affect the enzymatic cleavage as nucleotides in the vicinity of the AP site (−2, −1, +1, +2) participate in anchoring the flipped-out abasic nucleotide to the active site of the enzyme.³² However, our newly discovered +8 mismatch that is quite far from the AP site still inhibits the cutting effect of endonuclease IV. The possible reason may be conformational changes. The intrinsic mechanism is worth further study.The property newly found by us could be applied for constructing DNA sensing platforms with extremely high selectivity when a double mismatched strand is regarded as having single base mismatch in comparison to its corresponding single mismatch strand, which can be used for the study of single nucleotide polymorphisms (SNPs). As shown in Table S3,^† for +3(A:C)/+8 and +3(A:C) mismatched pairs, DFs ranged from 258.28 to 877.07 for all types of mismatches, which are remarkable values for genotyping.It is clear that our design does not exhibit requirements regarding the sequence of the target as we have found a mismatched pair having remarkable DFs for all types of mismatches. The fluorescence-quencher probe can be flexibly designed in accordance to the target strand. It can be an improvement and complement for the method developed by Xiao et al. For some specific sequence, the method may exhibit better performance than the method developed before. For non-targeted mutations, the positive rate reported using this assay is greatly reduced. Thus, our new findings improve the recognition library of Endo IV, covering a wider variety of mutations combined with other Endo-IV-based methods.