YAP/TAZ affects the development of pulmonary fibrosis by regulating multiple signaling pathways

Molecular and Cellular Biochemistry - Coronin-3 (coronin-1C), a homotrimer F-actin-binding protein, has been reported to be important for metastasis in several types of cancers such as lung cancer,...     

桐花树内生和根际细菌多样性及抗血栓活性研究

红树植物内生菌在红树共生体的物质循环、能量传递和健康维护等方面起着重要作用。为探究红树植物内生菌的多样性,进一步揭示内生菌在红树共生体的功能多样性提供菌种资源,该研究选择6种分离培养基和采用传统稀释涂布法对从广西北海滩涂上采集的桐花树组织和根际土壤样品进行分离,对获得的可培养细菌进行多样性分析,并通过体外溶栓实验筛选出具有抗血栓活性的菌株。结果表明:(1)基于16S rRNA基因序列系统进化分析,从桐花树组织和根际土壤中共获得125株细菌;分布于变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes) 3个门27个科39个属74个种中,芽孢杆菌属为优势菌属,菌株数量占13.5%。(2)抗血栓活性实验表明,初筛获得18株具有抗血栓活性细菌,总阳性率为24.32%;将初筛有活性的菌株进行复筛和重复验证实验,进一步验证其活性,结果复筛出3株细菌B1850、B1989和B2632具有很强抗血栓活性。综上所述,广西北海滩涂上红树植物桐花树中存在丰富的可培养细菌资源,具有从中挖掘新的纤溶酶和开发溶血栓药物的潜力。     

非结核分枝杆菌肺病临床分离株的菌种鉴定及临床特征分析

为探究非结核分枝杆菌（nontuberculous mycobacterium,NTM）肺病临床分离株的菌种分布及临床特征, 对2017年5月―2018年10月就诊于复旦大学附属中山医院的90例NTM肺病患者的样本进行分析。采用快速全自动分枝杆菌培养和药物敏感检测系统（BACTEC MGIT960 System）或改良罗氏培养法对90例患者的采集样本进行培养,利用基质辅助激光解析/电离飞行时间质谱（matrix assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS）进行菌种鉴定,并对回顾性分析收集的90例患者的临床资料进行分析。结果NTM菌种鉴定为9种,其中慢速生长分枝杆菌65例,以胞内分枝杆菌（54.4%,49/90）占多数;快速生长分枝杆菌25例,以脓肿分枝杆菌（22.2%,20/90）占多数。90例患者中确诊67例、疑似23例。确诊患者中少见菌种所占比例较低（6.0% vs 26.1%,P = 0.016）。确诊与疑似患者在临床表现方面未见显著差异,但确诊患者有抗NTM治疗史的比例显著高于疑似患者（85.1% vs 4.3%,P < 0.001）。确诊患者中,快速生长NTM肺病患者既往抗结核治疗史的比例显著高于慢速生长组（52.9% vs 24.0%,P = 0.036）。本研究结果为NTM肺病的临床诊治提供了数据参考。     

Parallel evolution of jugal structures in Devonian athyridide brachiopods

Here, we describe Sinathyris crassa gen. et sp. nov., a new early Emsian (Early Devonian) athyridide brachiopod with a double spiralium from the Guangxi Province of southern China. Unlike the majority of genera of the subfamily Helenathyridinae, which possess accessory spiral lamellae developed directly from the jugal branches, the form described here shows these lamellae arising from a distally bifurcating jugal stem. These differences suggest that the double spiralium in S. crassa might have appeared independently from the double spiralium of the helenathyridins. To test the subfamily assignment of Sinathyris gen. nov., we carried out phylogenetic analyses, which indicate that the new genus is more appropriately referred to the Didymothyridinae. The cladistic analyses of the athyridides indicate that double spiralia have developed independently among these brachiopods at least five times during their evolutionary history.     

志贺菌CRISPR的检测及其与耐药的关系

【目的】检测志贺菌成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR),并分析其与志贺菌耐药的关系。【方法】根据CRISPR DB数据库公布的志贺菌确定的CRISPR结构序列CRISPR-S2、CRISPR-S4和可能的CRISPR结构序列CRISPR-S1、CRISPR-S3设计四对引物,对60株志贺菌进行PCR扩增。采用CRISPR Finder分析CRISPR,采用改良K-B药敏纸片法检测志贺菌耐药情况,并分析CRISPR-S4与耐药的关系。【结果】确定的CRISPR结构的总阳性率为95%,四个CRISPR位点组成12种CRISPR谱型(A-L),除K型外均含确定的CRISPR结构,新发现1种重复序列和12种间隔序列。60株志贺菌的多重耐药率为53.33%。CRISPR-S4阳性菌株与阴性菌株之间,耐药的分布差异无统计学意义,但多重耐药菌株和耐TE菌株CRISPR-S4的重复序列多为R4.1,其3’末端缺失碱基AC;多重耐药菌株CRISPR-S4的间隔序列多为Sp5.1、Sp6.1和Sp7。【结论】CRISPR在志贺菌中广泛分布。CRISPR重复序列的变异和间隔序列的多样性可能与志贺菌耐药有关。     

响应面法优化蕤核叶片总黄酮提取工艺

采用超声波辅助提取的方法从蕤核叶片中提取总黄酮.利用响应面法(RSM法)研究了超声提取时间、乙醇浓度、液固比、提取温度等因素对总黄酮得率的影响,确定了超声波辅助提取蕤核叶片总黄酮的最佳工艺参数.结果表明,超声波辅助提取蕤核叶片总黄酮的最佳工艺务件为:超声时间50.1 min,乙醇浓度59.5%,液料比24.9:1,提取...     

Construction of a recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strain expressing a fusion protein of Omp22 and HpaA from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> for oral vaccine development

Objectives

To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.

Results

The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).

Conclusions

This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

     

In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye

The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K⁺], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K⁺ ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K⁺-selective, increasing >4-fold with increasing [K⁺] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K⁺] was 20.8 ± 0.3 mm and decreased by inhibition of the Na⁺/K⁺ pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR_inh-172), or K⁺ channels (TEA or XE991). ASL [K⁺] was increased by forskolin but not affected by Na⁺/K⁺/2Cl⁻ cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K⁺] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K⁺]. [K⁺] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K⁺] is not a factor in CF lung disease. In intact airways, ASL [K⁺] was also well above extracellular [K⁺]: 22 ± 1 mm in pig trachea ex vivo and 16 ± 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K⁺] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K⁺].The airway surface liquid (ASL)2 is the thin layer of aqueous fluid that lines the mucosal surface of the airways, forming the interface between airway epithelial cells and the gas phase. The ASL contains water, ions, and macromolecules. It is believed that ASL volume and composition are tightly regulated to maintain a nonviscous fluid layer for mucociliary clearance by underlying epithelial cells and to support the intrinsic antimicrobial function of defensins and other macromolecules (1, 2). Abnormalities in ASL volume and/or composition are proposed to be important in the pathogenesis of cystic fibrosis (CF) lung disease (3). The ASL is formed by a combination of fluid secretion by airway submucosal glands, convective fluid transport up the airway tree, and water/ion transport by airway surface epithelial cells, the latter likely playing a key role in active regulation of ASL volume and ionic composition.Although early data suggested that salt concentration in the ASL is low in normal airways (4), the current view is that the ASL is approximately isotonic in both normal and CF airways (5–7). A concern with older measurements of ASL composition involving fluid collection by filter paper or microcapillaries is perturbation of the airway surface and the sampling, by capillary forces, of more fluid than contained in the very thin (tens of microns) ASL layer. Studies using ion-sensitive microelectrodes, although technically demanding and requiring direct contact with the ASL, provided evidence for a nearly isotonic ASL (5). Our laboratory developed ratioable fluorescent dyes to measure ASL [Na⁺] and [Cl⁻], in which the ASL was fluorescently stained for determination of ion concentrations by ratio imaging microscopy (7). ASL salt concentration ([Na⁺] and [Cl⁻]) was found to be approximately isotonic in airway epithelial cell cultures, mouse trachea and small airways, and ex vivo human airways, without differences in CFTR deficiency (7, 8). We also found the ASL to be approximately isosmolar with serum using fluorescent, osmotically sensitive liposomes (9).Relatively little is known about ASL potassium concentration ([K⁺]) or its regulation. As diagrammed in Fig. 1A, transcellular transport of K⁺ is believed to involve the coordinated activities of a Na⁺/K⁺ pump, Na⁺/K⁺/2Cl⁻ cotransporter, and K⁺ channel(s) at the basolateral membrane and a H⁺/K⁺ pump and K⁺ channel(s) at the apical membrane. The airway epithelium also has significant paracellular ion permeability. There is evidence for functional expression of several types of K⁺ channels in cell lines derived from airways/lung, including Ca²⁺-activated, cAMP-activated, and voltage-activated K⁺ channels (10–13). Steady state [K⁺] in a stationary ASL (without fluid convection) should depend on the activities of cell membrane K⁺ pumps and ion transporters, as well as non-K⁺ ion channels, such as CFTR and ENaC, which are involved in establishing membrane potentials and thus the electrochemical driving forces for K⁺ transport.Open in a separate windowFIGURE 1.Cell model and perfusion chamber for measurements of ASL K⁺ concentration. A, schematic of airway epithelium showing principal ion transporters on the apical and basolateral plasma membranes, and paracellular pathway. B, schematic of perfusion chamber. Cells on a porous filter, facing upward, are imaged from above after fluorescent dye staining of the ASL. The under surface of the porous filter is perfused continuously. C, short circuit current (I_sc) in HBE cell monolayers in response to the additions of amiloride (10 μm), forskolin (10 μm), CFTR_inh-172 (10 μm), ATP (100 μm), and CaCC_inh-A01 (30 μm), a CaCC-specific inhibitor. D, transepithelial PD in response to amiloride, forskolin, CFTR_inh-172, ATP, and CaCC_inh-A01. The data in C and D are representative of four sets of measurements.The purpose of this study was to develop a noninvasive fluorescence method to measure [K⁺] in the ASL and to establish the major determinants of [K⁺] regulation. Following several years of synthetic chemistry, we developed a series of water-soluble K⁺ sensors, the first being TAC-Red, in which K⁺ binding to a triazacryptand (TAC) K⁺ ionophore results in fluorescence enhancement of a conjugated xanthylium chromophore by a charge transfer quenching mechanism (14). TAC-Red was used to follow K⁺ waves in the extracellular space in brain in a neuroexcitation model of cortical spreading depression. Second generation K⁺ sensors of different colors, TAC-Crimson (15) and TAC-Lime (16), work by a similar K⁺-sensing mechanism but utilize different chromophores. These indicators are selective for K⁺ under physiological conditions and respond to changes in [K⁺] in milliseconds or less (15). For the measurements here, we synthesized a dextran conjugate containing TAC-Lime, which has K⁺-sensitive green fluorescence, and tetramethylrhodamine, a reference chromophore with K⁺-insensitive red fluorescence. The indicator allowed technically straightforward determination of ASL [K⁺] in cell culture and in vivo models by fluorescence ratio imaging.