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.

     

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。     

小花老鼠簕可培养细菌多样性及其生物学活性研究

小花老鼠簕(Acanthus ebracteatus)是一种生长在红树生态系统的珍稀真红树植物,具有较高的药用价值。为研究小花老鼠簕内生及根际可培养细菌多样性,挖掘其潜在新物种及具有特殊生物学活性的菌株,该文利用7种不同培养基,通过传统稀释涂布法对小花老鼠簕各植物组织及根际土壤可培养细菌进行分离,基于16S rRNA基因序列解析其内生及根际细菌群落结构和多样性特征,应用植物病原菌平板对峙实验和平铺捕食活性测试分析其可培养细菌的抗菌活性。结果表明：(1)基于16S rRNA基因序列分析,发现从小花老鼠簕的根、茎、叶、花及根际土壤中分离得到144株可培养细菌,这些细菌隶属于18目26科37属66种,芽孢杆菌属(Bacillus)和链霉菌属(Streptomyces)为优势菌属,分别占细菌种数的15.1%和13.6%;(2)拮抗多种植物病原菌试验结果显示,获得29株具有拮抗植物病原菌活性的细菌,10株具有广谱抑菌活性,其中链霉菌属菌株拮抗作用最强且菌株Y129为潜在新物种。(3)捕食活性测试结果显示,有5株细菌对金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡...     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

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.     

北部湾南流江流域植被净初级生产力时空分布及其驱动因素

分析北部湾南流江流域净初级生产力时空动态变化特征及其驱动机制,为该区域生态环境保护及应对气候变化提供科学依据。基于光能利用率模型（CASA）,利用遥感数据、气象数据和植被类型数据估算了研究区2000-2015年流域的净初级生产力（NPP）,借助于Theil-Sen趋势、Mann-Kendall检验以及Hurst指数等数理统计方法对研究区NPP的时空变化特征、未来趋势及其驱动因素进行了定量化分析。结果表明：①时间尺度上,流域NPP总体上呈现出波动上升趋势,增速为44.03 g C m^-2（10 a）^-1,快于广西自治区,上游和下游地区NPP快于全区,而中游地区慢于全区;②空间尺度上,流域NPP的分布规律表现出明显的地域分异,中游最高（1098.99 g C/m²）,下游次之（1041.71 g C/m²）,而上游最小（1013.22 g C/m²）。NPP的Sen趋势度介于-77.10-74.80 g C m^-2 a^-1之间,在空间上呈现出增加的趋势。③空间波动性上,流域NPP的变异系数较大,其值介于0.01-0.71,其中洪潮江水库、小江水库周边以及玉林市的城乡建设用地扩张区域处于高波动状态,而流域中部的六万大山以及五皇山地带则处于低波动状态;④未来变化趋势上,流域NPP的Hurst指数范围介于0-0.99之间,平均值为0.70,呈现单峰右偏分布,预示着流域NPP未来处于持续性增加的趋势;⑤驱动机制上,流域NPP与多年平均气温呈正相关关系,与年均降水量呈负相关关系,气温是该流域植被NPP的主控因子。由耕地转化为建设用地所导致的NPP损失值最大,其值达到4715.62 t/a,而草地转换为建设用地导致NPP损失值最小,其值仅为184.63 t/a。     

Insect kinin mimics act as potential control agents for aphids: Structural modifications of Trp4

Insect kinins are endogenous, biologically active peptides with various physiological functions. The use of insect kinins in plant protection is being evaluated by many groups. Some kinins have been chosen as lead compounds for pest control. We previously reported an insect kinin mimic IV-3 that had insecticidal activity. And by introducing a strong electron withdrawing group (-CF₃) on the benzene ring (Phe²), we discovered a compound, L ₇ , with better activity than lead IV-3 . In this work, taking L ₇ as the lead compound, we designed and synthesized 13 compounds to evaluate the influence of position 4 (Trp⁴) of insect kinin on insecticidal activity, by replacing the H atom on tryptophan with -CH₃ and -Cl or substituting the indole ring of tryptophan with the benzene, naphthalene, pyridine, imidazole, cyclohexane, and alkyl carboxamides. The aphid bioassay results showed that the compounds M ₁ , M ₃ , and M ₅ were more active than the positive control, pymetrozine. Especially, replacing the side chain by an indole ring with 4-Cl substitution ( M ₁ , LC₅₀ = 0.0029 mmol/L) increased the aphicidal activity. The structure–activity relationships (SARs) indicated that the side chain benzene ring at this position may be important to the aphicidal activity. In addition, the toxicity prediction by Toxtree, and the toxicity experiments on Apis mellifera suggested that M ₁ was no toxicity risk on a non-target organism. It could be used as a selective and bee-friendly insecticide to control aphids.