军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

耐除草剂玉米G1105E-823C定性检测方法

转基因耐除草剂玉米G1105E-823C是经过改造的转mG2-aroA基因耐草甘膦玉米新品系,具有更高的草甘膦耐受性,目前已完成生产性试验,具有重要的产业化应用前景。但目前尚无针对G1105E-823C的转化体特异性检测方法的相关报道,这十分不利于对该品系的检测及监管。基于此,以G1105E-823C转化体特异性序列为靶标,建立了转基因耐除草剂玉米G1105E-823C的普通PCR和实时荧光PCR定性检测方法。结果表明,2种方法均能检测出转基因耐除草剂玉米G1105E-823C转化体成分,且具有较高的特异性。普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。研究建立的2种定性检测方法为转基因耐除草剂玉米G1105E-823C的精准检测提供了新的技术手段,可为农业转基因监管提供技术支撑。     

Sex identification of morphologically-undifferentiated Siberian sturgeon with statistical analysis of gene expression patterns

The purpose of this study was to detail a simple strategy for sexing morphologically-undifferentiated fish using statistical analysis of gene expression patterns characterized by quantitative PCR. This approach is especially relevant for species without known genomic sex markers. The method was developed for early identification of female Siberian sturgeon as part of a genomics study. That study documented activation of the enzyme 17ß-hydroxy-steroid-dehydrogenase (hsd17b1) in future ovarian tissue at 3 months of age, concurrent with a small forkhead box L2 (foxl2) peak and emerging cytochrome P450, family 19, subfamily A (cyp19a1) expression. Major cyp19a1 and foxl2 peaks occurred in presumptive female gonads at 5–6 months. This pattern suggested a genetic relay mediating estrogen production throughout differentiation, possibly to maintain gonadal femininity. Genes involved in stem cell proliferation (lim homeobox 2 (lhx2)) and somatic-germ cell interaction maintenance (iroquois homeobox 5 (irx5) and iroquois homeobox 3 (irx3)) were also expressed during molecular differentiation, at 5–6 months. The roles of lhx2, irx3, and irx5 in fish sex differentiation should be confirmed using other methodologies. These results indicate that estrogens are crucial for ovarian differentiation in basal non-teleost fish, consistent with well-established patterns in teleosts, with hsd17b1 as one of the earliest biomarkers of gonadal development.     

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii

The resistance of Gram-negative bacteria to β-lactam antibiotics stems mainly from β-lactamase proteins that hydrolytically deactivate the β-lactams. Of particular concern are the β-lactamases that can deactivate a class of β-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by β-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D β-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the “real-time” deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a β-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or β-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling β-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.     

熊去氧胆酸联合非诺贝特对原发性胆汁性胆管炎的临床疗效及安全性

摘要 目的：分析熊去氧胆酸联合非诺贝特对原发性胆汁性胆管炎无熊去氧胆酸生化反应的临床疗效及安全性。方法：151例原发性胆汁性胆管炎无熊去氧胆酸患者按随机数表法分为73例对照组和78例研究组。对照组在常规治疗基础上给予安慰剂联合熊去氧胆酸治疗,研究组在常规基础上给予非诺贝特联合熊去氧胆酸治疗,两组均持续治疗12个月。比较两组临床疗效,肝功能,血脂指标,肝纤维化指标,免疫球蛋白G（IgG）、免疫球蛋白M（IgM）,瘙痒及乏力评分,不良反应发生情况。结果：治疗后,研究组总有效率高于对照组,比较差异有统计学意义（P<0.05）。治疗后,两组肝功能指标均降低,研究组低于对照组,比较有统计学意义（P<0.05）。治疗后,两组总胆固醇（TC）、甘油三酯（TG）均降低,研究组低于对照组,比较有统计学意义（P<0.05）,两组治疗前后低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）比较无统计学意义（P>0.05）。治疗后,两组肝纤维化指标均降低,研究组低于对照组,比较有统计学意义（P<0.05）。治疗后,两组IgG、IgM均降低,研究组低于对照组,比较有统计学意义（P<0.05）。治疗后,两组瘙痒、乏力评分均降低,研究组低于对照组,比较有统计学意义（P<0.05）。两组不良反应发生率比较差异无统计学意义（P>0.05）。结论：熊去氧胆酸联合非诺贝特对原发性胆汁性胆管炎无熊去氧胆酸生化反应的疗效较好,能够改善肝功能,且未明显增加药物不良反应。     

冬虫夏草菌实时荧光定量PCR内参基因的筛选

以冬虫夏草单子囊孢子分离得到的菌株TZ8-1的3种菌丝形态为实验材料,提取RNA,经反转录获取cDNA,选择了11个持家基因为候选内参基因（18S rRNA、APRTase、β-TUB、RPL2、EF1-α、PGI、PGM、H⁺-ATPase、ACT1、UBQ和GAPDH）,根据该菌基因组注释结果来设计引物,采用实时荧光定量PCR（qRT-PCR）技术进行定量扩增,利用geNorm、NormFinder和BestKeeper算法程序进行表达稳定性评估,并用RefFinder对评估结果进行综合排比,最终筛选得到了最适内参基因。结果表明,所选取的11个候选内参基因均可作为冬虫夏草菌菌丝体时期的内参基因,稳定性最好的3个内参基因分别是UBQ、PGE和ACT1,稳定性较差的3个内参基因分别是GAPDH、RPL2和β-TUB。     

Unweighted state as a sidestep preparation improve the initiation and reaching performance for basketball players

The preparatory motion of a defensive motion in contact sport such as basketball should be small and involve landing on both feet for strict time and motion constraints. We thus proposed the movement creating a unweighted state. Ten basketball players performed a choice reaction sidestepping task with and without the voluntary, continuous vertical fluctuation movement. The results indicated that the preparatory movement shortened the time of their sidestep initiation (301 vs. 314 ms, p = 0.011) and reaching performance (883 vs. 910 ms, p = 0.018) but did not increase their peak ground reaction force or movement velocity. The mechanism of the improvement was estimated to be the following: in the preparation phase, the vertical body fluctuation created the force fluctuation; after the direction signal, the unweighted state can shorten the time required to initiate the sidestepping (unweighted: 279 ms; weighted: 322 ms, p = 0.002); around the initiation phase, the dropping down of the body and weighted state can contribute to the reaching performance. We conducted additional experiment investigating muscle–tendon-complex dynamics and muscle activity using ultrasound device and electromyography. The result suggests that the building up of active state of muscle might explain the improvement of sidestepping performance.