Structural determinants of tobacco vein mottling virus protease substrate specificity

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac‐RETVRFQSD) at 1.7‐Å resolution. As observed in several crystal structures of TEV protease, the C‐terminus (～20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ～10‐fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k_cat and K_m for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.     

DRR1 is expressed in the developing nervous system and downregulated during neuroblastoma carcinogenesis

Down-regulated in renal cell carcinoma 1 (DRR1) is mapped at 3p21.1, and is a candidate tumor suppressor gene. However, its biological roles have yet to be elucidated. Here, we developed polyclonal antibodies against DRR1 protein, and examined its expression during embryogenesis and carcinogenesis. The DRR1 protein was preferentially expressed in axonal projections of the central and peripheral nervous system of mice during embryonic days 10.5-16.5. Consistent with this expression pattern, the protein was detected in the neurites of primary cultured cortical neurons of rats at embryonic day 18.5. Survival of these cells was significantly inhibited by RNAi-induced downregulation of DRR1 expression. DRR1 was poorly expressed in established cancer cell lines, including neuroblastoma cells, whereas strong expression was observed in normal cells. A neuroblastoma model, MYCN transgenic mice, revealed that DRR1 protein was expressed in the celiac ganglion 2 weeks after birth when neuroblast hyperplasia was also observed; however, there was no longer any expression of DRR1 protein in tumors originating from the ganglion 8 weeks after birth. Together, our data indicate that DRR1 protein is expressed in normal cells, particularly in the nervous system during embryogenesis, is involved in neuronal cell survival, and is downregulated during neuroblastoma carcinogenesis.     

实时影像融合的超声虚拟导航技术联合射频消融术治疗原发性肝癌合并门静脉癌栓患者的疗效及对血清Bax、Cyfra21-1的影响

摘要 目的：探讨实时影像融合的超声虚拟导航技术联合射频消融术治疗原发性肝癌合并门静脉癌栓患者的疗效及对血清BCL-2同源的水溶性相关蛋白（Bax）、细胞角蛋白19片段（Cyfra21-1）的影响。方法：选择本院2017年1月到2021年4月收治的原发性肝癌合并门静脉癌栓患者82例作为研究对象,根据1：1随机数字表法将患者分为虚拟导航组与对照组各41例,虚拟导航组给予实时影像融合的超声虚拟导航技术联合射频消融术治疗,对照组给予单纯超声引导联合射频消融术治疗。结果：虚拟导航组的进针次数、融合时间、布针时间少于对照组（P<0.05）;虚拟导航组治疗后3个月的胆汁瘤、肝脓肿、膈肌损伤、肺部感染等并发症发生率为4.9 %,低于对照组的29.3 %（P<0.05）。虚拟导航组治疗后3个月的总有效率为82.9 %,高于对照组的51.2 %（P<0.05）。两组治疗后3个月的血清谷丙转氨酶（ALT）、谷草转氨酶（AST）水平低于治疗前,虚拟导航组低于对照组（P<0.05）。两组治疗后的血清Bax、Cyfra21-1含量低于治疗前,虚拟导航组低于对照组（P<0.05）。结论：实时影像融合的超声虚拟导航技术联合射频消融术治疗原发性肝癌合并门静脉癌栓能降低血清Bax、Cyfra21-1含量,改善患者的肝功能,提高消融效率,还可减少并发症的发生,最终提高患者的总体治疗效果。     

一项小鼠采血新技术——颌下静脉丛采血法

目的介绍小鼠颌下静脉丛采血方法。方法选用2月龄昆明小鼠,固定小鼠后,将采血注射针头刺入颌下静脉丛血管取血。结果单人操作约1 min内可完成小鼠颌下静脉丛采血,采血量达到0.3-0.5 mL。结论此方法无需麻醉、创伤小、采血量大并可重复采血,是一种简便、安全、快速、采血量多的采血方法。     

Leaf economics and hydraulic traits are decoupled in five species‐rich tropical‐subtropical forests

Leaf economics and hydraulic traits are critical to leaf photosynthesis, yet it is debated whether these two sets of traits vary in a fully coordinated manner or there is room for independent variation. Here, we tested the relationship between leaf economics traits, including leaf nitrogen concentration and leaf dry mass per area, and leaf hydraulic traits including stomatal density and vein density in five tropical‐subtropical forests. Surprisingly, these two suites of traits were statistically decoupled. This decoupling suggests that independent trait dimensions exist within a leaf, with leaf economics dimension corresponding to light capture and tissue longevity, and the hydraulic dimension to water‐use and leaf temperature maintenance. Clearly, leaf economics and hydraulic traits can vary independently, thus allowing for more possible plant trait combinations. Compared with a single trait dimension, multiple trait dimensions may better enable species adaptations to multifarious niche dimensions, promote diverse plant strategies and facilitate species coexistence.     

大鼠左肾静脉狭窄致左肾淤血性损伤模型的建立

目的观察大鼠左肾静脉不同程度狭窄所致左肾病变,为实验研究左肾静脉受压综合征肾组织淤血性损伤提供合适的动物模型。方法采用左肾静脉不全结扎的方法建立大鼠左肾静脉狭窄模型。将大鼠分为4组,假手术组和左肾静脉狭窄1.0mm模型组、0.7mm模型组、0.5mm模型组。术后7周处死动物。肾组织行病理学检查。肾皮质匀浆检测丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果病理学检查见1.0mm模型组未见明显病变,0.7mm和0.5mm模型组肾小球系膜区增生,小管、间质细胞浸润和纤维化形成,0.5mm模型组病变程度较重。各模型组左肾皮质丙二醛含量均显著增高,超氧化物歧化酶活性均显著降低,变化幅度随狭窄程度的增大而增大。结论大鼠左肾静脉狭窄程度为0.7mm时,各项观察指标与胡桃夹综合征（NCS）患者的临床实际情况最接近,而1.0mm和0.5mm相对偏轻和偏重,0.7mm模型组可以作为大鼠左肾静脉狭窄致左肾淤血实验研究的合适模型。     

Effects of long-term serial cell passaging on cell spreading,migration, and cell-surface ultrastructures of cultured vascular endothelial cells

The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.     

关节置换术后抗凝药物预防深静脉血栓及相关并发症的临床研究进展

抗凝药物有助于预防全髋关节置换术和全膝关节置换术后深静脉血栓形成,临床上最常使用的传统抗凝药物如低分子肝素、华法林等可以起到很好的预防效果。目前有一类新的口服抗凝药物已经用于临床,为关节置换术后患者带来了一种更方便、安全和有效预防血栓的治疗选择。本篇综述主要针对传统抗凝药物低分子肝素及维生素K拮抗剂,直接凝血酶抑制剂达比加群,以及选择性Xa因子抑制剂利伐沙班和阿哌沙班,对迄今为止传统抗凝药物在全髋关节置换术和全膝关节置换术患者中的临床使用经验、优缺点、以及新型口服抗凝药物最新临床用药进展进行综述,为关节置换术后患者预防深静脉血栓提供用药参考。     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Higher level of plasma bioactive molecule sphingosine 1-phosphate in women is associated with estrogen

Both sphingosine 1-phosphate (S1P) and estrogen have been documented to play endothelial protective roles. However, it remains unclear whether estrogen could regulate the anabolism of the bioactive molecule S1P and the underlying mechanisms. In this study, 108 healthy participants were separated into three age groups, and their plasma S1P levels were analyzed by liquid chromatography tandem mass spectrometry. Results showed that the plasma S1P levels were significantly higher in women than those in men within the age of 16–55 years old and higher in pre-menopausal than post-menopausal women. The experiment in C57 BL/6 mice confirmed the gender difference of plasma S1P level. In vitro study demonstrated that after the stimulation of 17β-estradiol (E2), S1P levels both in EA.hy926 cells and the culture media were increased about 9 and 3 times, respectively; the mRNA expression, the protein level and the activity of sphingosine kinase (SphK) 1, not SphK2, were markedly increased; the mRNA and protein expression of ATP-binding cassette transporter (ABC) C1, G2 and S1P transporter spinster homolog 2 (Spns2) were significantly elevated; furthermore, the mRNA and protein expressions of S1P receptors (S1PRs) 1–2 were increased in a time-dependent manner. This study suggests that E2 markedly improves S1P synthesis by activating SphK1 and induces S1P export via activating ABCC1, G2 and Spns2 from endothelium system, which may consequently lead to the gender difference of plasma S1P in adult human and mouse. The results of this study suggest that E2 may exert its vasculoprotective function by activation of the SphK1–S1P–S1PR signaling axis.