Molecular mechanisms of An-Chuan Granule for the treatment of asthma based on a network pharmacology approach and experimental validation

An-Chuan Granule (ACG), a traditional Chinese medicine (TCM) formula, is an effective treatment for asthma but its pharmacological mechanism remains poorly understood. In the present study, network pharmacology was applied to explore the potential mechanism of ACG in the treatment of asthma. The tumor necrosis factor (TNF), Toll-like receptor (TLR), and Th17 cell differentiation-related, nucleotide-binding oligomerization domain (NOD)-like receptor, and NF-kappaB pathways were identified as the most significant signaling pathways involved in the therapeutic effect of ACG on asthma. A mouse asthma model was established using ovalbumin (OVA) to verify the effect of ACG and the underlying mechanism. The results showed that ACG treatment not only attenuated the clinical symptoms, but also reduced inflammatory cell infiltration, mucus secretion and MUC5AC production in lung tissue of asthmatic mice. In addition, ACG treatment notably decreased the inflammatory cell numbers in bronchoalveolar lavage fluid (BALF) and the levels of pro-inflammatory cytokines (including IL-6, IL-17, IL-23, TNF-alpha, IL-1beta and TGF-beta) in lung tissue of asthmatic mice. In addition, ACG treatment remarkably down-regulated the expression of TLR4, p-P65, NLRP3, Caspase-1 and adenosquamous carcinoma (ASC) in lung tissue. Further, ACG treatment decreased the expression of receptor-related orphan receptor (RORγt) in lung tissue but increased that of Forkhead box (Foxp3). In conclusion, the above results demonstrate that ACG alleviates the severity of asthma in a ´multi-compound and multi-target’ manner, which provides a basis for better understanding of the application of ACG in the treatment of asthma.     

一氧化氮参与盐胁迫下长春花酚类代谢的调控研究

为研究外源一氧化氮(NO)调控盐胁迫下长春花中酚类化合物的响应,采用液相色谱—质谱联用(LCMS)技术靶向分析梯度浓度硝普纳(SNP)处理对盐胁迫下长春花幼苗根、茎、花、叶4个部位中酚类化合物组分及含量水平的变化。结果共鉴定出L-苯丙氨酸和18种酚类物质,C6C1类5种、C6C3类5种、C6C3C6类8种,其中原儿茶酸、绿原酸、槲皮素在长春花根、茎、花、叶4个部位中均存在;不同浓度SNP处理后长春花不同部位酚类化合物响应积累明显不同,其中C6C1和C6C3小分子酚酸类化合物主要积累在根和茎中,C3C6C3类主要富集在花和叶中;L-苯丙氨酸在茎、叶中相对含量较高,盐胁迫下茎中含量显著升高,且随外源NO浓度增大呈下降趋势。外源NO影响盐胁迫下植物器官中酚类化合物的积累和变化,其中根和茎响应敏感,从种类和相对含量的角度,茎和叶更适合检测酚类化合物。     

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally, from B.1.17 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) to B.1.1.529 (Omicron). The emerging or reemerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics, pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 cells. We utilized both animal models of human ACE2 (hACE2) transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.     

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.     

隆肛蛙属种群形态量度分析

对隆肛蛙属Feirana中隆肛蛙F.quadrana和太行隆肛蛙F.taihangnicus的15个种群565只标本的28项形态性状进行了测量,并运用典型判别分析法对其进行分析,分析结果表明:1)太行隆肛蛙和隆肛蛙的形态差异明显,形态度量信息支持太行隆肛蛙与隆肛蛙是不同的物种;2)原隆肛蛙河南伏牛山种群和山西中条山种群均为太行隆肛蛙的地理种群;3)隆肛蛙的种群间形态差异明显,其中四川安县种群、陕西周至种群和湖北利川种群与模式产地重庆巫山种群的差异可能达到了亚种或亚种以上分化水平.     

群体成员大小差异对不同生境鲤科鱼类集群行为的影响

为研究群体成员大小差异对不同喜好生境鱼类集群行为特征的影响, 实验分别以鳊(Parabramis pekinensis)和中华倒刺鲃(Spinibarbus sinensis)幼鱼为实验对象, 比较分析4尾等大小(E)和不等大小(2大2小, NE)实验鱼群体的自发游泳速度、空间分布以及对恐吓刺激反应等集群行为参数的差异。结果显示: (1)和鳊相比, 中华倒刺鲃有更高的自发游泳速度、速度同步性和排列方向的极性, 但二者对恐吓刺激的反应率及反应的协调一致性相似; (2)当群体成员大小出现差异时, 两种鱼群体排列方向的极性不受影响, 且大小个体成员间的速度及其同步性均没有差异, 但整体的速度同步性与等大小群体相比有所下降; (3)个体间距离数据显示, 个体大小差异不会影响两种鱼群体的凝聚力; (4)群体成员在两种鱼群中偏好位置不同, 当群体成员大小不同时, 大个体成员更偏好占据领头鱼位置; (5)群体成员大小的差异导致两种鱼对刺激的反应率下降。研究表明: 中华倒刺鲃具有更高的活跃性、更好的群体运动的协调性, 可能与其流水生境相关; 当群体成员大小出现差异时, 成员不分大小在整体上协调运动的速度和方向, 并保持群体有较高的凝聚力, 但两种鱼类自发游泳速度调整策略截然不同(鳊大小个体速度妥协趋同, 而中华倒刺鲃低速个体速度提高); 群体成员大小差异导致鱼群对恐吓刺激的反应率有所下降, 可能原因包括体形差异导致的社会因素造成敏锐性下降、信息交流效率受阻和(或)集群收益代价出现分化影响一致决策的形成等。     

细胞表面的物理化学修饰

移植排斥是生物学中的重大问题,其理论涉及众多生物学科,包括免疫学、生物化学、分子生物学、生理学、细胞生物学、实验血液学等;其实践涉及同种和异种细胞、组织、器官移植,关系到肿瘤、糖尿病、急性放射病、免疫缺陷症、器官衰竭等数以百万计患者的治疗和健康。使用化学材料聚乙二醇、海藻酸钠、壳聚糖、多聚赖氨酸等,发挥化学、物理、生物学科交叉的优势,在移植物细胞表面进行物理化学反应,修饰和改造细胞表面抗原分子,有望为克服移植排斥反应开辟新的途径。     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

小鼠孤雌胚胎干细胞的建立及其向运动神经元分化的初探

文章采用小鼠的孤雌囊胚建立胚胎干细胞系,探究其向运动神经元分化的可能,为临床治疗以及研究基因组印记与神经分化的的关系提供理论基础。结果表明:卵母细胞孤雌激活率达到93.26%,成功建立了8个孤雌胚胎干细胞系,建系率达到23.53%。克隆表达多潜能标记Oct4及细胞表面标记SSEA-1,有高水平的碱性磷酸酶活性,在细胞第10代和第30代时核型分析检测显示为正常的40条染色体。体内、外均分化出三胚层来源的细胞。联合应用全反式维甲酸(RA)、音猬因子(Shh)及细胞外基质,小鼠孤雌胚胎干细胞可被诱导表达运动神经元的标志性标记HB9、Olig2。