Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely used for structural characterization of bacterial endotoxins (lipid A). However, the mass spectrometric behavior of the lipid A molecule is highly dependent on the matrix. Furthermore, this dependence is strongly linked to phosphorylation patterns. Using lipid A from Escherichia coli O116 as a model system, we have investigated the effects of different matrices and comatrix compounds on the analysis of lipid A. In this paper, we report a highly sensitive matrix system for lipid A analysis, which consists of 5-chloro-2-mercaptobenzothiazole matrix and EDTA ammonium salt comatrix. This matrix system enhances the sensitivity of the analysis of diphosphorylated lipid A species by more than 100-fold and in addition provides tolerance to high concentrations of sodium dodecyl sulfate (SDS) and tolerance to sodium chloride and calcium chloride at 10 μM, 100 μM, and 10 μM concentrations. The method was further evaluated for analysis of lipid A species with different phosphorylation patterns and from different bacteria, including Helicobacter pylori, Salmonella enterica serovar Riogrande, and Francisella novicida.Lipopolysaccharide (LPS) is a major component of the outer membranes of Gram-negative bacteria (21). Typically, LPS molecules consist of a hydrophilic carbohydrate portion and a hydrophobic lipid A (or endotoxin). The lipid A molecule consists of a fatty acyl substituted β-d-GlcN-(1-6)-α-GlcN disaccharide unit that usually carries phosphate groups. Diphosphorylated lipid A is generally presumed to be phosphorylated at C-1 and C-4′ positions (9); however, lipid A moieties containing pyrophosphate (PP) groups have also been reported (13). The presence of phosphate groups in lipid A greatly affects the endotoxic properties of LPS (22). Deletion of either of these groups reduces an endotoxic activity of the resulting monophosphorylated LPS by approximately 100-fold (18). For example, monophosphorylated lipid A has been used as an adjuvant in a hepatitis B vaccine in Europe (1, 12).Mass spectrometry (MS) has been widely used to gain information about the heterogeneity, i.e., the number of different species of lipid A families and a distribution of fatty acids on each glucosamine residue (2, 3, 9, 16, 20, 23, 28, 29, 30, 32, 35, 36). Detailed structural information, including the phosphorylation pattern of lipid A, can be obtained by tandem mass spectrometry. Several matrices have been used for the analysis of lipid A using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and 6-aza-2-thiothymine (ATT) (8). Although DHB has been widely used for peptide analysis, it produces uneven crystals and leads to poor spot-to-spot reproducibility (3, 6, 11). Furthermore, the low solubility in the solvent compatible with lipid A and nonuniformity in a matrix layer (crystals) can lead to variations in the ionization yield across the sample resulting in formation of “hot” (or “sweet”) spots (14). On the other hand, 5-chloro-2-mercaptobenzothiazole (CMBT) was found to offer excellent spot-to-spot reproducibility because of the homogeneous crystallization of the analyte/matrix mixture over the sample spot (33). CMBT is soluble in methanol-chloroform-water (4:4:1, vol:vol:vol), a solvent compatible with lipid A molecules, especially hexaacylated species. Thus, it has been widely used for lipid A analysis (4, 9, 23, 35, 33). Interestingly, different preparation procedures for analysis of lipid A species dictate a selection of the preferred matrix system (10). For example, lipid A prepared using a TRI Reagent-based procedure with a CMBT matrix was preferable for the detection of phosphoethanolamine modifications (35). On the other hand, the analysis of lipid A prepared using an LPS extraction kit-based procedure with DHB was preferable for the detection of aminoarabinose modification (10). In addition, divalent cations, such as Ca²⁺ or Mg²⁺, can bridge the phosphorylated negatively charged groups between neighboring LPS molecules to form aggregates (24). Thus, there is a need for technologies capable of characterizing lipid A from biologically relevant samples in an accurate, rapid, and highly sensitive manner. Here we attempt to establish an optimized MALDI MS matrix system for the sensitive analysis of lipid A, especially its diphosphorylated forms, including both pyrophosphorylated and bisphosphorylated species. We also propose to incorporate a complex reagent (additive or comatrix) for reducing the interference of cations (5, 7, 15).     

Expanded Multilocus Sequence Typing and Comparative Genomic Hybridization of Campylobacter coli Isolates from Multiple Hosts

The purpose of this work was to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with certain host species. Genotyping and ClonalFrame analyses of an expanded 16-gene multilocus sequence typing (MLST) data set involving 85 isolates from 4 different hosts species tentatively supported the development of C. coli host-preferred groups and suggested that recombination has played various roles in their diversification; however, geography could not be excluded as a contributing factor underlying the history of some of the groups. Population genetic analyses of the C. coli pubMLST database by use of STRUCTURE suggested that isolates from swine form a relatively homogeneous genetic group, that chicken and human isolates show considerable genetic overlap, that isolates from ducks and wild birds have similarity with environmental water samples and that turkey isolates have a connection with human infection similar to that observed for chickens. Analysis of molecular variance (AMOVA) was performed on these same data and suggested that host species was a significant factor in explaining genetic variation and that macrogeography (North America, Europe, and the United Kingdom) was not. The microarray comparative genomic hybridization data suggested that there were combinations of genes more commonly associated with isolates derived from particular hosts and, combined with the results on evolutionary history, suggest that this is due to a combination of common ancestry in some cases and lateral gene transfer in others.Campylobacter species are a leading bacterial cause of gastroenteritis within the United States and throughout much of the rest of the developed world. According to the CDC, there are an estimated 2 million to 4 million cases of Campylobacter illness each year in the United States (37). Campylobacter jejuni is generally recognized as the predominant cause of campylobacteriosis, responsible for approximately 90% of reported cases, while the majority of the remainder are caused by the closely related sister species Campylobacter coli (27). Not surprisingly, therefore, the majority of research on Campylobacter has centered on C. jejuni, and C. coli is a less studied organism.A multilocus sequence typing (MLST) scheme of C. jejuni was first developed by Dingle et al. (13) on the basis of the genome sequence of C. jejuni NCTC 11168. There have also been a number of studies using the genome sequence data to develop microarrays for gene presence/absence determination across strains of C. jejuni and to identify the core genome components for the species (6, 15, 32, 33, 42, 43, 53, 57). Although C. coli is responsible for fewer food-borne illnesses than C. jejuni, the impact of C. coli is still substantial, and there is also evidence that C. coli may carry higher levels of resistance to some antibiotics (1). C. coli and C. jejuni also tend to differ in their relative prevalences in animal host species and various environmental sources (4, 48, 58), and there is some evidence that both taxa may include groups of host-specific putative ecotype strains (7, 36, 38, 39, 52, 56). At present, there is only a single draft genome sequence available for C. coli, and there are no microarray comparative genomic hybridization data for C. coli strains. Thus, there is no information on intraspecies variability in gene presence/absence in C. coli and how such variability might correlate with host species.The purpose of this work was to develop and apply an expanded 16-locus MLST genotyping scheme to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with isolates derived from different host species.     

OsZIP5 is a plasma membrane zinc transporter in rice

Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice.     

Growth and photosynthetic efficiency promotion of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) by endophytic bacteria

Very little is known about the physiological interactions between plants and endophytic bacteria. We investigated the impact of three endophytic bacteria, Bacillus pumilus 2-1, Chryseobacterium indologene 2-2, and Acinetobacter johnsonii 3-1, on the photosynthetic capacity and growth of sugar beet. Endophyte-free plants were obtained first and infected with the bacteria. Measurements of total chlorophyll content revealed very significant differences between endophyte-free beet plants and some infected by endophytic bacteria. The maximum photochemical yield (Fv/Fm) was used to determine any photosynthetic effect on plants caused by biotic or abiotic factors. After 30 days of growth, there was significantly higher Fv/Fm for endophyte-infected than endophyte-free plants. The light response curves of beet showed that photosynthetic capacity was significantly increased in endophyte-infected plants. Photosynthesis of endophyte-free plants was saturated at 1,300 μmol m⁻² s⁻¹, whereas endophyte-infected plants were not saturated at the irradiance used. The effect seemed to be due to promotion of electron transport in the thylakoid membranes. Promotion of photosynthetic capacity in sugar beet was due to increased chlorophyll content, leading to a consequent increased carbohydrate synthesis. It is possible that the increased maximum yield of photosynthesis in sugar beet was promoted by phytohormones and produced by the bacteria.     

Single intravenous injection of plasmid DNA encoding human paraoxonase-1 inhibits hyperlipidemia in rats

Paraoxonase-1 (PON1, EC 3.1.8.1) is a high-density lipoprotein (HDL)-associated antioxidant enzyme, and its activity correlates negatively with the level of plasma low-density lipoprotein cholesterol (LDL-C) and triglyceridemia (TG). In this study, we examined the therapeutic effect of plasmid DNA containing the human PON1 gene (pcDNA/PON1) in hyperlipidemic model rats. The rats were fed a high-fat and high-cholesterol diet for 25 days to produce a hyperlipidemic animal model. Single intravenous injection of pcDNA/PON1 into model rats prevented dyslipidemia and hepatic lipid accumulation. The mechanisms of pcDNA/PON1 in treating hyperlipidemia were associated with increases of serum antioxidant PON1 and SOD activities, and with reduction of the levels of total cholesterol (TC), LDL-C and TG. The results suggest the potential therapeutic effect of pcDNA/PON1 on hyperlipidemia.     

Association Analysis of the RET Proto-Oncogene with Hirschsprung Disease in the Han Chinese Population of Southeastern China

Hirschsprung disease (HSCR) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. This study investigated a possible role of the RET proto-oncogene in sporadic HSCR patients in the Han Chinese population. Our results indicated that rs1800858, rs1800860, rs1800863, and rs2075912, located in exons 2, 7, 15, and intron 19 of RET, are strongly associated with the disease (P < 0.01), with rs1800860 and rs1800863 playing a protective role in the pathogenesis of HSCR in the Chinese population. We also showed that the haplotype consisting of four SNPs is significantly associated with HSCR. We did not find a significant difference in the CA-repeat in intron 5 of RET between cases and controls. Our study provided further evidence that the RET gene is involved in the susceptibility to HSCR in the Han Chinese population.     

变形链球菌细胞壁对EAhy926细胞TLR4、IL-6和IL-8表达的影响

【目的】观察变形链球菌细胞壁对EAhy926细胞的增殖活性、TLR4的表达和炎性细胞因子IL-6和IL-8分泌的影响,初步探讨变形链球菌致血管内皮细胞TLR4表达、炎症反应及其两者之间的关系。【方法】变形链球菌细胞壁作用于EAhy926细胞,MTT法检测EAhy926细胞的增殖活性;RT-PCR法检测EAhy926细胞TLR4、IL-6和IL-8 mRNA的表达;流式细胞术检测EAhy926细胞表面TLR4的表达;细胞生物活性方法和ELISA分别检测EAhy926细胞IL-6和IL-8的分泌;抗体阻断实验观察IL-6和IL-8的表达与TLR4的关系。【结果】不同浓度的变形链球菌细胞壁作用6 h可促进EAhy926细胞的增殖(P0.05),但12 h后可明显抑制该细胞的生长(P0.05),呈现显著的时间和剂量依赖性。变形链球菌细胞壁作用于EAhy926细胞后,TLR4 mRNA和蛋白水平的表达量随着作用时间延长而逐渐增高,在16 h达到高峰,24 h后又逐渐下降(P0.01);IL-6和IL-8的表达也呈现明显的时间依赖性增高(P0.05)。经TLR4抗体阻断后,变形链球菌细胞壁刺激EAhy926细胞IL-6和IL-8的产生明显减少(P0.01)。【结论】变形链球菌细胞壁可明显抑制EAhy926细胞的生长,上调该细胞TLR4的表达,促进炎性细胞因子IL-6和IL-8的分泌;IL-6和IL-8的产生与TLR4的表达上调密切相关。     

厌氧氨氧化菌富集培养物对羟胺的转化研究

【目的】羟胺是厌氧氨氧化的重要中间产物,本研究旨在探明厌氧氨氧化菌对羟胺的转化特性。【方法】采用厌氧氨氧化菌富集培养物,以羟胺和亚硝酸盐为基质进行分批培养试验,检测反应液中基质和产物的消涨情况。【结果】不接种厌氧氨氧化富集培养物时,羟胺和亚硝酸盐具有化学稳定性,彼此不发生化学反应;接种厌氧氨氧化富集培养物后,羟胺和亚硝酸盐发生化学反应;反应过程中有中间产物氨的产生和转化,最大氨氮积累浓度为0.338mmol/L;液相中总氮浓度从起始的4.694mmol/L降至结束时的0.812mmol/L,转化率为82.7%。羟胺和亚硝氮浓度均为2.5mmol/L时,羟胺最大比污泥转化速率为0.535mmol/(gVSS.h),是厌氧氨氧化反应体系中氨氮最大比污泥转化速率的1.81倍。将羟胺浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高26.7%和120.7%,最大氨氮积累浓度为0.795mmol/L;将亚硝氮浓度提高至5.0mmol/L时,羟胺和亚硝氮转化速率分别提高6.9%和9.0%,最大氨氮积累浓度为1.810mmol/L。【结论】厌氧氨氧化富集培养物能够转化羟胺,其对羟胺的转化速率高于对氨的转化速率。羟胺相对过量可显著加快羟胺和亚硝酸盐的转化速率,亚硝酸盐相对过量对羟胺和亚硝氮转化速率影响不大,提高羟胺或亚硝氮浓度均会增大中间产物氨氮的积累。实验现象可用van de Graaf模型解释,对于进一步开发厌氧氨氧化工艺具有重要的理论意义。     

一株假单胞菌(Pseudomonas alcaliphila MBR)好氧还原亚硒酸钠为红色单质硒

【目的】本实验室保藏的一株异化硝酸盐还原菌(Pseudomonas alcaliphila MBR),其能够在好氧环境下以有机碳源为电子供体,把易溶解、高毒性亚硒酸钠还原成为红色单质硒,本文对该菌株还原亚硒酸盐的特征进行了研究。【结果】结果表明该菌株可以在pH为6-11环境中生长,对亚硒酸钠有较强抗性,其MIC(minimal inhibitory concentration)可高达50 mmol/L。在5天时间内,菌体以柠檬酸钠为电子供体,把2 mmol/L亚硒酸钠完全还原为红色单质硒并主要积累于胞外。硝酸盐和还原型谷胱甘肽对菌体还原亚硒酸钠具有促进作用,初步确定菌体对亚硒酸钠的还原是细胞膜或细胞质中的某些物质催化的结果。【结论】本项研究为应用Pseudomonas alcaliphila MBR于生物反应器提供了重要基础。     

醉马草内生菌的分离、鉴定及杀虫效果

【目的】探明醉马草内生菌的种类,筛选对农作物虫害有毒杀作用的菌株。【方法】采用研磨法从健康醉马草植物的根、茎、叶和种子中进行菌种分离;通过对其形态、培养特征、生理生化及其他生物学特性的研究;16SrDNA及ITS序列的系统发育学分析进行鉴定;用玻片浸渍法和喷雾法筛选产杀虫活性物质菌株。【结果】获得细菌89株,分别属于枯草芽孢杆菌属(Bacillus)、链霉菌属(Streptomyces)、棒状杆菌属(Corynebacterium)、叶杆菌属(Phyllobacterium)、鞘脂单胞菌属(Sphingomonnas)、类芽孢杆菌(Paenibacillus)、假单胞菌属(Pseudomonas)和不动杆菌属(Acinetobacter)8个属;真菌2株,分别属于麦角菌属(Claviceps)和毛壳菌属(Chaetomium)。经初筛及复筛,内生菌娄彻氏链霉菌Streptomyces rochei(GA)和黑麦麦角菌Claviceps purpurea(PF-2)发酵液粗提物对棉蚜虫(Aphis gossypii)致死率达85%以上。【结论】醉马草内生菌株PF-2和GA的粗提代谢物对棉蚜虫有明显的毒杀作用,为开发新的生物源农药提供了生物源物质。