Efficient gene silencing induction in tomato by a viral satellite DNA vector

Virus-induced gene silencing (VIGS) in tomato (Lycopersicon esculentum Mill.) is currently routinely analysed using Tobacco rattle virus (TRV)-based vector. We recently reported a new vector system modified from DNA beta (DNAm beta) of Tomato yellow leaf curl China virus (TYLCCNV) for VIGS analysis in Solanaceous species including tomato. Here, we describe DNAm beta-induced gene silencing in tomato. We found that DNAm beta-induced gene silencing was initiated from vascular tissues, and later scattered to other tissues. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants, was expressed in a variety of tissues and organs including leaf, shoot, stem, flower and fruit, and could be achieved at any growth stage. It was insensitive to temperature as high as 32 degrees C and no symptoms were observed in silenced plants. The DNAm beta vector worked efficiently in at least seven tomato cultivars, indicating that this system has great potential as a versatile VIGS system for routine functional analysis of genes in tomato.     

Association of HLA genes with ankylosing spondylitis in Han population of eastern China

Ankylosing spondylitis (AS) is a chronic inflammatory disorder with a multifactorial genetic basis. HLA-B27 was reported with the greatest susceptibility to AS but did not act alone. The aim of this study was to search for other gene(s) associated with AS independently of HLA-B27 using 13 microsatellite markers spanning 1.5 Mb from locus TAP1 to HLA-Cw and a single-nucleotide polymorphism marker within NFkappaBIL1 gene promoter. Genotyping for microsatellites was performed in 175 AS patients of eastern Chinese and 219 ethnically matched healthy controls using polymerase chain reaction with fluorescence-labelled primers, whereas the SNP marker was genotyped by direct DNA sequencing. Allele as well as haplotype frequencies were compared between cases and controls, and a linkage disequilibrium analysis was performed to estimate the LD relationship between the candidate regions. The frequencies of alleles D6S2811*128, STR_MICA*A5.1 and D6S2672*109, as well as haplotypes D6S2811*128-D6S2927*213-D6S2810*340, D6S2927* 221-D6S2810*350-MICA*A5.1, and D6S2810*350-MICA*A5.1-D6S2800* 136 were significantly increased in B27-positive AS patients when compared with B27-positive controls. The results indicated that there may be other gene(s) within the HLA region, especially around locus HLA-B or HLA-Cw, with susceptibility to AS independently of HLA-B27.     

局限性胃肠道间质瘤危险程度分级标准的应用与评价

目的 探讨Fletcher和Miettinen两种分级标准对局限性胃肠道间质瘤（GIST）危险程度的评估效能。方法 收集220例有完整临床病理及随访资料的局限性GIST,应用Fletcher和Miettinen两种分级标准对肿瘤的危险程度进行评估,生存分析比较两种标准在预测肿瘤恶性潜能中的意义。结果 该组资料按照Fletcher分级标准进行评估,高危组的总生存率和术后无复发、无转移生存率（无病生存率）显著低于极低、低和中危三组,但极低、低和中危三组之间总生存率和无病生存率差异无统计学意义。在高危组中,小肠和直肠GIST的总生存率和无病生存率显著低于胃GIST;而在中危组中,小肠GIST的无病生存率显著低于胃GIST。该组资料按照Miettinen分级标准进行评估,高危组的总生存率和无病生存率显著低于极低、低和中危三组,但极低、低和中危三组之间总生存率和无病生存率差异无统计学意义。在极低、低、中和高危各个危险组中,胃、小肠和直肠GIST之间总生存率及无病生存率差异均无统计学意义。结论 Fletcher分级标准简明易用,但Miettinen分级标准按不同发病部位评估危险程度能更准确地认识局限性GIST的生物学行为,对正确选择高危患者进行靶向辅助治疗具有重要参考价值。     

培养细胞免疫细胞化学技术的特点与临床应用

培养细胞免疫化学技术作为一种跨领域的研究手段,是将免疫细胞化学技术与细胞培养技术密切结合的技术,可获得单纯从体内实验难以达到的效果。培养细胞免疫组织化学技术与组织切片的免疫组织化学技术基本原理和操作是相同的,但又有许多要特别注意的细节。[第一段]     

Characterization of endogenous betaine γ-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells

Increase in mRNA expression and transport activity of the betaine γ-amino-n-butyric acid cotransporter (BGAT) in response to hyperosmolality has been previously shown in MDCK cells. However, the hyperosmolality-induced response of endogenous BGAT protein expression was not investigated in detail. We show two forms of endogenous BGAT immunoreactivity that are expressed in MDCK II cells. Both are sensitive to Peptide N-Glycosidase F (PNGase F), suggesting that they are N-glycosylated proteins. One band, about 75 kDa, is resistant to Endo H, while the other 55 kDa band is sensitive to it, suggesting that they are fully N-glycosylated mature form in the post-Golgi compartment and core-glycosylated immature form in the endoplasmic reticulum (ER), respectively. When treated with hyperosmolality, they are significantly increased. But the rate of BGAT processing, as assessed by the ratio of mature to immature form, is not increased, suggesting that hyperosmolality does not facilitate the export of BGAT from the ER to the secretory pathway. Surface biotinylation and confocal microscopy show that hyperosmolality significantly increases the amount of the mature form of BGAT on the basolateral membrane with a very small fraction on the apical membrane. We conclude that BGAT is an N-glycosylated protein with two glycoforms and endogenous BGAT synthesis rather than processing is involved in the adaptation to the hyperosmotic stress. Xue-Mei Zhang and Xi-Tao Wang contributed equally to this work.     

Epidural spinal cord stimulation plus quipazine administration enable stepping in complete spinal adult rats

We hypothesized that epidural spinal cord stimulation (ES) and quipazine (a serotonergic agonist) modulates the excitability of flexor and extensor related intraspinal neural networks in qualitatively unique, but complementary, ways to facilitate locomotion in spinal cord-injured rats. To test this hypothesis, we stimulated (40 Hz) the S(1) spinal segment before and after quipazine administration (0.3 mg/kg, ip) in bipedally step-trained and nontrained, adult, complete spinal (mid-thoracic) rats. The stepping pattern of these rats was compared with control rats. At the stimulation levels used, stepping was elicited only when the hindlimbs were placed on a moving treadmill. In nontrained rats, the stepping induced by ES and quipazine administration was non-weight bearing, and the cycle period was shorter than in controls. In contrast, the stepping induced by ES and quipazine in step-trained rats was highly coordinated with clear plantar foot placement and partial weight bearing. The effect of ES and quipazine on EMG burst amplitude and duration was greater in flexor than extensor motor pools. Using fast Fourier transformation analysis of EMG bursts during ES, we observed one dominant peak at 40 Hz in the medial gastrocnemius (ankle extensor), whereas there was less of dominant spectral peak in the tibialis anterior (ankle flexor). We suggest that these frequency distributions reflect amplitude modulation of predominantly monosynaptic potentials in the extensor and predominantly polysynaptic pathways in the flexor muscle. Quipazine potentiated the amplitude of these responses. The data suggest that there are fundamental differences in the circuitry that generates flexion and extension during locomotion.     

qPCR快速检测金黄色葡萄球菌及MRSA方法的建立及评价

目的建立q PCR法快速检测金黄色葡萄球菌及其mec A基因,以期快速精准诊断金黄色葡萄球菌感染及初步判断耐药情况。方法从NCBI数据库下载金黄色葡萄球菌nuc、atl、ica B、fnb A、hla、srap基因序列用于鉴定标志筛选,mec A基因序列用于MRSA标志筛选;经DNA MAN比对后,选择各基因保守区分别设计1~2套引物、探针,建立单重及双重q PCR,用临床分离株及标准菌株筛选出检测性能最好的基因片段作为检测标志物,建立金黄色葡萄球菌鉴定和耐药双重q PCR检测方法,并进行性能评价。结果经筛选,金黄色葡萄球菌atl基因(CP009361.1:1010217~1010341)、mec A基因(KF058908.1:1715~1843)两片段检测性能最优,将其作为标志物建立双重q PCR法。该方法的检测下限均低至4 copies/反应;扩增线性范围均达2.0×102~8copies/m L。335份金黄色葡萄球菌(含94份MRSA)培养阳性的患者样本中,q PCR法分别检出SA 335份,MRSA 94份;95份金黄色葡萄球菌培养阴性的患者样本中,q PCR法分别检出SA 17份,MRSA 4份,经PCR产物测序,与标准菌株同源性均≥90%。双重q PCR法从样品处理到报告结果≤2.0 h。结论 q PCR法方法简便、快速、灵敏度高、特异性好,可望提高金黄色葡萄球菌感染的诊断能力并实现快速检测,为尽早精准治疗赢得时间。     

Membrane Loaded Copper Oleate PEGylated Liposome Combined with Disulfiram for Improving Synergistic Antitumor Effect <Emphasis Type="BoldItalic">In Vivo</Emphasis>

Purpose

This work aims to create a novel Cu²⁺ liposome with excellent loading stability and develop synergistic effect with disulfiram (DSF) for the treatment of tumor.

Methods

Copper oleate was incorporated into the liposome membrane via alcohol injection method in this work. In vitro release test was applied to evaluate the release profile of the liposomes. Pharmacokinetic studies were performed in rats and the antitumor efficacy was assessed in mice bearing hepatoma xenografts.

Results

The copper oleate liposome (Cu(OI)₂-L) was formulated and the loading efficiency were more than 85%. TEM images confirmed that the Cu(OI)₂-L had a spherical morphology with an average diameter of 100 nm. Cu(OI)₂-L displayed a biphasic release profile, with >70% retained drug over 8 h incubation in PBS at pH 7.4. Pharmacokinetic studies demonstrated that Cu(OI)₂-L had a prolonged circulation time and increased AUC when compared to the injection of copper oleate solution. The antitumor efficacy test demonstrated an enhanced tumor inhibition rate with the treatment of Cu(OI)₂-L and DSF nanoparticles, indicating an improved synergistic antitumor effect.

Conclusions

The Cu(OI)₂-L was suitable to be employed in combination with disulfiram for tumor treatment and can also open up opportunities for targeted delivery of copper.

     

Genistein-loaded poloxamer 403/407 mixed micelles: preparation and pharmacokinetic study in rats

In the present study, we aimed to prepare poloxamer 403/407 mixed micelles in order to improve the solubility and oral bioavailability of genistein.Genistein was incorporated in the mixed poloxamer micelles by thin-film hydration method, and its physicochemical properties, including particle size, zeta potential, entrapment efficiency and drug loading, were investigated.In vitro release of genistein from the mixed micelles was monitored by dialysis method, and pharmacokinetic study of genistein loaded mixed micelles was carried out in rats. We found that the particle size and zeta potential of mixed micelles were (20.31±0.43) nm and (–8.94±0.35) mV, with encapsulation efficiency 90.59%±0.67% and drug loading 7.74%±0.05%. Solubility of genistein in mixed micelles reached 3.80 mg/mL, which was about 130 times higher than that in water.Genistein-loaded mixed micelles showed sustained release characteristics in vitro with no burst release phenomenon, but it was faster than suspension.The AUC_0–t andAUC_0–∞ of mixed micelles were 196.74% and 204.62% greater than that of genisein suspension, respectively.Consequently,poloxamer 403/407 mixed micelles significantly improved the solubility and oral bioavailability of genistein, which could be used as an effective drug delivery system for oral administration of poorly soluble drugs.     

国内部分医院β-内酰胺类抗生素皮肤试验现状调查分析

摘 要 目的：采用问卷调查了解并分析我国医院β-内酰胺类抗生素皮肤试验的实际操作情况。方法：通过电子邮件或传真方式对国内100家医院的临床药师进行问卷调查。问卷内容涵盖医院的名称、级别;β-内酰胺类药物皮肤试验方法,包括皮试液的来源和浓度、操作方法、观察时间、阳性结果的判定标准;涉及过敏史时,β-内酰胺类药物的皮肤试验以及针对口服青霉素制剂是否需要皮肤试验等14个方面的相关问题。结果：共收到信息完整的反馈问卷80份（反馈比80%）。调查显示各医院β-内酰胺类药物皮肤试验流程各有不同。不同医院在抗生素皮试品种、皮试方法、涉及过敏史时药物的选择以及口服青霉素制剂是否进行皮试等方面均存在明显差异。结论：本研究反映了现阶段我国医疗机构在β-内酰胺类抗生素皮肤试验方面缺乏统一标准,亟需尽快出台相关临床指南或共识,以保证临床使用该类药物的安全性与规范性。