Semi-perfusion cultures of suspension MDCK cells enable high cell concentrations and efficient influenza A virus production

Control and prevention of rapid influenza spread among humans depend on the availability of efficient and safe seasonal and pandemic vaccines, made primarily from inactivated influenza virus particles. Current influenza virus production processes rely heavily on embryonated chicken eggs or on cell culture as substrate for virus propagation. Today’s efforts towards process intensification in animal cell culture could innovate viral vaccine manufacturing using high-yield suspension cells in high cell density perfusion processes. In this work, we present a MDCK cell line adapted to grow as single cell suspension with a doubling time of less than 20 h, achieving cell concentrations over 1 × 10⁷ cells/mL in batch mode. Influenza A virus titer obtained in batch infections were 3.6 log₁₀(HAU/100 µL) for total- and 10⁹ virions/mL for infectious virus particles (TCID₅₀), respectively. In semi-perfusion mode concentrations up to 6 × 10⁷ cells/mL, accumulated virus titer of 4.5 log₁₀(HAU/100 µL) and infectious titer of almost 10¹⁰ virions/mL (TCID₅₀) were possible. This exceeds results reported previously for cell culture-based influenza virus propagation by far and suggests perfusion cultures as the preferred method in viral vaccine manufacturing.     

胸腔镜肺小结节切除术前微弹簧圈定位的临床应用研究

目的:研究孤立性肺结节(SPN)胸腔镜术前CT引导下双弹簧圈精准标记定位的应用价值。方法:回顾分析43例SPN胸腔镜术前定位病例资料，包括双弹簧圈组22例，Hook-wire定位组21例。统计双弹簧圈定位的术中、术后并发症，衔接期时间以及作楔形切除所用时间，并将两组结果进行对比分析。结果:两组病例定位均取得成功；双弹簧圈组的气胸发生率(9.0%)，肺出血发生率(9.0%)，胸痛发生率(9.0%)均低于Hook-wire组，其中肺出血发生率与Hook-wire组比较，差异有统计学意义(P＜0.01)；衔接时间双弹簧圈组（15.38±8.32）h长于Hook-wire组（4.21±3.29）h，差异有统计学意义（P＜0.05）；作楔形切除所用时间双弹簧圈组(21.01±7.14)min与Hook-wire组(18.22±5.18)min差异无统计学意义(P＞0.05)。结论:采用双微弹簧圈进行SPN胸腔镜手术前精准标记定位安全可靠、效果良好，与Hook-wire定位比较并发症发生率更低，并可获得更长的衔接期，具有较高的应用价值。     

A highly tumor-targeted nanoparticle of podophyllotoxin penetrated tumor core and regressed multidrug resistant tumors

Podophyllotoxin (PPT) exhibited significant activity against P-glycoprotein mediated multidrug resistant (MDR) tumor cell lines; however, due to its poor solubility and high toxicity, PPT cannot be dosed systemically, preventing its clinical use for MDR cancer. We developed a nanoparticle dosage form of PPT by covalently conjugating PPT and polyethylene glycol (PEG) with acetylated carboxymethyl cellulose (CMC-Ac) using one-pot esterification chemistry. The polymer conjugates self-assembled into nanoparticles (NPs) of variable sizes (20–120 nm) depending on the PPT-to-PEG molar ratio (2–20). The conjugate with a low PPT/PEG molar ratio of 2 yielded NPs with a mean diameter of 20 nm and released PPT at ∼5%/day in serum, while conjugates with increased PPT/PEG ratios (5 and 20) produced bigger particles (30 nm and 120 nm respectively) that displayed slower drug release (∼2.5%/day and ∼1%/day respectively). The 20 nm particles exhibited 2- to 5-fold enhanced cell killing potency and 5- to 20-fold increased tumor delivery compared to the larger NPs. The biodistribution of the 20 nm PPT-NPs was highly selective to the tumor with 8-fold higher accumulation than all other examined tissues, while the larger PPT-NPs (30 and 120 nm) exhibited increased liver uptake. Within the tumor, >90% of the 20 nm PPT-NPs penetrated to the hypovascular core, while the larger particles were largely restricted in the hypervascular periphery. The 20 nm PPT-NPs displayed significantly improved efficacy against MDR tumors in mice compared to the larger PPT-NPs, native PPT and the standard taxane chemotherapies, with minimal toxicity.     

Discovery of nitazoxanide-based derivatives as autophagy activators for the treatment of Alzheimer's disease

Drug repurposing is an efficient strategy for new drug discovery. Our latest study found that nitazoxanide (NTZ), an approved anti-parasite drug, was an autophagy activator and could alleviate the symptom of Alzheimer's disease (AD). In order to further improve the efficacy and discover new chemical entities, a series of NTZ-based derivatives were designed, synthesized, and evaluated as autophagy activator against AD. All compounds were screened by the inhibition of phosphorylation of p70S6K, which was the direct substrate of mammalian target of rapamycin (mTOR) and its phosphorylation level could reflect the mTOR-dependent autophagy level. Among these analogs, compound 22 exhibited excellent potency in promoting β-amyloid (Aβ) clearance, inhibiting tau phosphorylation, as well as stimulating autophagy both in vitro and in vivo. What's more, 22 could effectively improve the memory and cognitive impairments in APP/PS1 transgenic AD model mice. These results demonstrated that 22 was a potential candidate for the treatment of AD.     

Graphene oxide-induced neurotoxicity on neurotransmitters,AFD neurons and locomotive behavior in Caenorhabditis elegans

Graphene oxide (GO) and graphene-based nanomaterials have been widely applied in recent years, but their potential health risk and neurotoxic potentials remain poorly understood. In this study, neurotoxic potential of GO and its underlying molecular and cellular mechanism were investigated using the nematode, Caenorhabditis elegans. Deposition of GO in the head region and increased reactive oxygen species (ROS) was observed in C. elegans after exposure to GO. The neurotoxic potential of GO was then investigated, focusing on neurotransmitters contents and neuronal activity using AFD sensory neurons. The contents of all neurotransmitters, such as, tyrosine, tryptophan, dopamine, tyramine, and GABA, decreased significantly by GO exposure. Decreased fluorescence of Pgcy-8:GFP, a marker of AFD sensory neuron, by GO exposure suggested GO could cause neuronal damage on AFD neuron. GO exposure led decreased expression of ttx-1 and ceh-14, genes required for the function of AFD neurons also confirmed possible detrimental effect of GO to AFD neuron. To understand physiological meaning of AFD neuronal damage by GO exposure, locomotive behavior was then investigated in wild-type as well as in loss-of-function mutants of ttx-1 and ceh-14. GO exposure significantly altered locomotor behavior markers, such as, speed, acceleration, stop time, etc., in wild-type C. elegans, which were mostly rescued in AFD neuron mutants. The present study suggested the GO possesses neurotoxic potential, especially on neurotransmitters and AFD neuron in C. elegans. These findings provide useful information to understand the neurotoxic potential of GO and other graphene-based nanomaterials, which will guide their safe application.     

倾向性评分匹配法评价冠心病支架置入术后再狭窄

目的引入倾向性评分匹配法(PSM)比较伴糖尿病的冠心病患者与单纯冠心病患者支架置入术后再狭窄的差异。方法回顾性分析在本院接受冠状动脉支架植入术患者资料,伴糖尿病的冠心病患者137例(CHD+DM组),单纯冠心病283例(CHD组)。使用SPSS软件的PSM功能对两组采用1∶1最邻近匹配法,得到组间协变量均衡的样本。匹配后的样本采用Cox比例风险模型评估支架术后再狭窄的危险因素。结果两组匹配成功各120例。Cox比例风险模型多因素分析,吸烟史(HR=2.50,95%CI:1.34~4.64,P=0.004)、高血压史(HR=2.24,95%CI:1.08~4.63,P=0.030)、肌酐清除率110ml/min(HR=3.12,95%CI:1.22~5.03,P=0.024)、冠状动脉多支病变(HR=2.15,95%CI:1.14~4.07,P=0.018)和伴糖尿病(HR=2.22,95%CI:1.14~4.33,P=0.020)是冠心病支架术后再狭窄的独立危险因素。PSM后,CHD+DM组1、3、5年的累计狭窄率分别为10.70%,40.30%和43.80%,CHD组为4.70%,23.70%和29.60%,差异有统计学意义(P=0.023)。结论应用PSM可以有效地均衡非随机研究组间的协变量,糖尿病是冠心病患者支架置入术再狭窄的危险因素。     

上海地区216例青少年年龄与颈椎骨成熟度关系的研究

目的:探讨青少年年龄与颈椎骨成熟度间的相关性。方法:采用双盲法,对216例10～20岁青少年的X线头颅侧位定位片,采用SAS6.12统计软件分析C2～C5颈椎体生长发育的状态,与年龄间的相互关系以及性别间有无差异。结果:随着年龄增长,C2～C5颈椎骨形态呈规律性变化,女性比男性约提前2a,在11岁和12岁年龄段有统计学差异,但形态变化无性别差异。青春快速生长发育期的开始时间和高峰期时间,个体差异大。结论:用头颅侧位片观察颈椎骨的形态变化判断儿童的生长发育状态,比用年龄来判断快速生长期更为准确,是临床上简便可行的方法。