电感耦合等离子体质谱在细菌多组分分析中的应用

对细菌特征蛋白和核酸进行高通量定量分析,有助于快速鉴定细菌种群及其功能研究。近年来,结合元素标记策略的电感耦合等离子体质谱（ICP-MS）在细菌蛋白和核酸检测中的应用受到关注。ICP-MS检测蛋白质、核酸等生物分子时,具有灵敏度高、线性范围宽、生物基体干扰小等优点,适用于细菌多组分定量分析。本文综述了细菌不同特征物质作为目标待测物的ICP-MS分析方法,并对其发展方向进行了展望,希望为细菌分析方法的研究提供参考。     

Review on microbial metabolomics of probiotics and pathogens: Methodologies and applications

In recent years, microbial metabolomics, a new field that has attracted wide attention, provides a map of metabolic pathways and clarifies the interaction mechanism between microorganisms and hosts. Many microorganisms are found in the human intestine, oral cavity, vagina, etc. Probiotics could maintain the good health of the host, while pathogens and an imbalance of bacterial flora lead to a series of diseases of the body and mind. Metabolomics is a science for qualitative and quantitative analysis of all metabolites in an organism or biological system, which could provide key information to understand the related metabolic pathways and associated changes. This approach analyzes the final products of cellular regulatory processes, the level of which can be regarded as the ultimate response of the biological system to genetic or environmental changes. Microbial metabolomics has been widely used in different research fields, such as microbial phenotypic classification, mutant screening, metabolic pathways, microbial metabolic engineering, fermentation engineering monitoring and optimization, microbial environmental pollution, and so on. However, there are only a few reviews on microbial metabolomics of probiotics and pathogens. This review summarizes the main methodologies, including sample preparation, identification of metabolites, data processing, and analysis. Recent applications in microbial metabolomics of probiotics and pathogens are also described. This paper first summarized the research progress and application of microbial metabolomics from two aspects: probiotics and pathogenic bacteria. Probiotics and pathogenic bacteria do not exist independently most of the time; hence, these were reviewed in the research field of coexistence of probiotics and pathogenic bacteria, which was subdivided into important microbial research fields closely related to human health, including the human gut, oral cavity, food, and nutrition-related microorganisms. Then, the main problems and trends associated with microbial metabolomics are discussed.     

Non-classical use of clathrin during bacterial infections

How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, E-cadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell-cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin-E-cadherin pathway, and for the entry of other bacteria that enter by a 'zipper' mechanism. By contrast, the clathrin-mediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion system and enter by the so-called trigger mechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells, i.e. internalization of objects much larger than previously accepted.     

Proteomics in gram-negative bacterial outer membrane vesicles

Gram-negative bacteria constitutively secrete outer membrane vesicles (OMVs) into the extracellular milieu. Recent research in this area has revealed that OMVs may act as intercellular communicasomes in polyspecies communities by enhancing bacterial survival and pathogenesis in hosts. However, the mechanisms of vesicle formation and the pathophysiological roles of OMVs have not been clearly defined. While it is obvious that mass spectrometry-based proteomics offers great opportunities for improving our knowledge of bacterial OMVs, limited proteomic data are available for OMVs. The present review aims to give an overview of the previous biochemical, biological, and proteomic studies in the emerging field of bacterial OMVs, and to give future directions for high-throughput and comparative proteomic studies of OMVs that originate from diverse Gram-negative bacteria under various environmental conditions. This article will hopefully stimulate further efforts to construct a comprehensive proteome database of bacterial OMVs that will help us not only to elucidate the biogenesis and functions of OMVs but also to develop diagnostic tools, vaccines, and antibiotics effective against pathogenic bacteria.     

Mycobacterium Avium-intracellulare complex infections in the acquired immunodeficiency syndrome

This reviw examines an important bacterial infection in acquired immunodeficiency syndrome (AIDS). Despite occasional infections with bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, and Nocardia in patients with AIDS, the primary problems of AIDS and invading bacterial infections center around mycobacteropsos. A unique feature of AIDS has been the common identification of disseminated infections with Mycobacterium avium-intracellulare. The following discussion examines our present understading of this group of organisms and how they interact with the compromised host.     

MALDI-TOF mass spectrometry of bacteria

The development of MALDI-TOF mass spectrometry methods for the characterization of bacteria is reviewed and discussed. The general use of MALDI for the characterization of large biomolecules led directly to obvious applications involving the analysis of isolated bacterial proteins. More surprising was the observation that MALDI-TOF mass spectrometry could be applied directly to crude cellular fractions or cellular suspensions and that the resulting data from such complex mixtures could provide evidence for chemotaxonomic classification. Versatility and the rapidity of analysis led to the rapid development of a number of MALDI-TOF methods involving bacteria. Examples of some of the applications covered in this review are the analysis of bacterial RNA and DNA, the detection of recombinant proteins, the characterization of targeted or unknown proteins, bacterial proteomics, the detection of virulence markers, and the very rapid characterization of bacteria at the genus, species, and strain level. The demonstrated capability of taxonomic classification at the strain level, using unprocessed cells, opens the possibility that MALDI-TOF and similar mass spectrometry approaches may contribute significantly to fulfilling emerging needs for the development of near real-time methods for the characterization of bacteria.     

Progresses of mycobacteriophage-based <i>Mycobacterium tuberculosis</i> detection

Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide, particularly in developing countries. A rapid and efficient method for TB diagnosis is indispensable to check the trend of tuberculosis expansion. The emergence of drug-resistant bacteria has increased the challenge of rapid drug resistance tests. Due to its high specificity and sensitivity, bacteriophage-based diagnosis is intensively pursued. In this review, we mainly described mycobacteriophage-based diagnosis in TB detection, especially two prevalent approaches: fluorescent reporter phage and phage amplified biologically assay (PhaB). The rationale of reporter phage is that phage carrying fluorescent genes can infect host bacteria specifically. Phage amplified biological assay based on the principle that phages can infect the live Mycobacterium tuberculosis< in the specimen under suitable conditions and produce plaques. Other phage-based diagnostic methods, such as a combination of the amplified biologically assay and nucleic acid amplification or lateral flow assays, are also actively explored. This review will help us improve the understanding of mycobacteriophages in TB detection and better promote the development of the rapid diagnosis of M. tuberculosis.     

Comparative ultrastructure of nonwounded Mexican lime and Yuzu leaves infected with the citrus canker bacterium Xanthomonas citri pv. citri

Ultrastructural aspects of citrus canker development were investigated in nonwounded leaves of citrus species by transmission electron microscopy (TEM). A susceptible species Mexican lime and a resistant species Yuzu were spray-inoculated with a virulent strain of Xanthomonas citri pv. citri. Initial symptoms occurred on Mexican lime ∼9 days after inoculation, whereas they appeared on Yuzu mostly 11 days after inoculation. In Mexican lime leaves, the bacterial invasion was usually accompanied by host cell wall dissolution and cellular disruption. Fibrillar materials from degenerated cell walls were usually found in intercellular spaces. Damaged host cells with necrotic cytoplasm showed the localized separation of plasma membrane from the cell wall. Bacterial multiplication and electron-transparent capsule-like structures around bacteria were commonly observed. Meanwhile, cell wall protuberances were prominent outside host cell walls in response to bacterial invasion in Yuzu leaves. Occlusion of intercellular spaces was also formed by the fusion of two or more individual cell wall protuberances originated from two adjacent host cells. Papillae-like materials accumulated locally within host cells in close proximity to bacteria. Some bacteria were found to be undergoing degeneration in xylem vessels. Also, the shrunken, inactive bacteria were surrounded by electron-translucent fibrillar materials in intercellular spaces, implying bacterial immobilization. These cellular responses are thought to be the consequences of defense responses of Yuzu leaves to invading bacteria. In both citrus species, X. citri pv. citri contained polyphosphate bodies showing electron-dense and elliptical structures in cytoplasm. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Bacteria-hemocyte interactions and phagocytosis in marine bivalves

Marine bivalves (such as mussels, oysters, and clams) are widespread mollusks in coastal waters at different latitudes; due to their filter-feeding habits, they accumulate large numbers of bacteria from the harvesting waters and may act as passive carriers of human pathogens. To cope with this challenge, bivalves possess both humoral and cellular defense mechanisms with remarkably effective capabilities. The circulating cells, or hemocytes, are primarily responsible for defense against parasites and pathogens; microbial killing results from the combined action of the phagocytic process with humoral defense factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), toxic oxygen intermediates, and various antimicrobial peptides. In this work, current knowledge of the mechanisms underlying the interactions between bacteria and the hemolymph components of marine bivalves is summarized. Bacterial susceptibility to hemolymph killing in different bivalve species may be a consequence of the different ability of bacterial products to attract phagocytes, the presence or absence of specific opsonizing molecules, the hemocyte capability to bind and engulf different bacteria, and the different bacterial sensitivity to intracellular killing. The role of soluble (e.g., agglutinins and opsonins) and surface-bound factors in bacterial phagocytosis by hemocytes of the most common marine bivalve species is described and the possibility that environmental temperatures and other seasonal factors may influence this process is considered. Moreover, the potential strategies used by bacteria to evade phagocytic killing by hemocytes are discussed. From the available data it is clear that several questions need further investigation; the elucidation of the factors influencing phagocytosis in bivalves and the fundamental strategies used by bacteria to escape hemolymph killing are important not only to understand bivalve immune defenses but also to explain the persistence of pathogenic bacteria in bivalve tissues and to predict the consequent impact on human health.     

Imaging of lactic acid bacteria with AFM--elasticity and adhesion maps and their relationship to biological and structural data

The adhesion of lactic acid bacteria to the intestinal epithelium is one of the most important factors determining probiotic ability of a bacterial strain. Studying bacterial adhesion requires knowledge of the structure and properties of the bacterial surface, which can be studied by atomic force microscopy under native conditions. The observation of the surface topography of bacteria from the species Lactobacillus crispatus, L. helveticus and L. johnsonii shows major differences between bacteria having a crystalline-like protein layer as part of the cell wall and those without such layers. Force volume images calculated into elasticity and adhesion force maps of different bacterial strains show that L. crispatus and L. helveticus have a surface with a homogeneous stiffness with no adhesion events. This is most likely caused by the S-layer, which completely covers the surface of the bacteria. We infer that the absence of adhesion peaks is caused by the semi-crystalline character of such protein layers, in agreement with the results obtained from electron microscopy. Analysis of a number of L. johnsonii strains shows that these bacteria have surface properties which strongly differ from the L. crispatus and L. helveticus strains. For L. johnsonii DMS20533 and L. johnsonii ATCC33200 high adhesion forces are observed, which can be related to a surface rich in polysaccharides. L. johnsonii ATCC332 has lower adhesion forces compared to the other two and, furthermore, the surface topography shows depressions. We suppose that this strain has a surface pattern consisting of crystalline-like proteins alternating with polysaccharide-rich domains. The wide variety in surface properties of lactobacilli could well have wide-ranging implications for food processing and for health benefits.     

Proteome-wide screening for the analysis of protein targeting of <i>Chlamydia pneumoniae</i> in endoplasmic reticulum of host cells and their possible implication in lung cancer development

Available reports have confirmed a link between bacterial infection and the progression of different types of cancers, including colon, lungs, and prostate cancer. Here we report the Chlamydia pneumonia proteins targeting in endoplasmic reticulum (ER) using in-silico approaches and their possible role in lung cancer etiology. We predicted 48 proteins that target human ER, which may be associated with protein folding and protein-protein interactions during infection. The results showed C. pneumoniae proteins targeting human ER and their implications in lung cancer growth. These targeted proteins may be involved in competitive interactions between host and bacterial proteins, which may change the usual pathway functions and trigger the development of lung cancer. Moreover, C. pneumoniae unfolded protein accumulation in the human ER possibly induces ER stress, consequently activating the unfolded protein response (UPR), and providing a favorable microenvironment for cancer growth. The current study showed the C. pneumoniae protein targeting in ER of host cell and their implication in lung cancer growth. These results may help researchers better manage lung cancer and establish a molecular mechanism for C. pneumoniae lung cancer association.     

基于LVQ网络算法的目标分类方法

随着机器人技术的不断发展,构建未知空间的环境地图以成为移动机器人技术中具有挑战性的研究课题.自主环境建模体现了机器人的感知能力和智能水平,在实际应用中具有十分重要的意义.文章研究了学习矢量量化算法在智能机器人对环境障碍物识别中的应用以及噪声、振幅、声纳TOF数据的偏移、距离以及目标体(锐角和边角)的角度等因素对系统分类性能的影响.实验表明,该方法对上述各种影响因素具备一定的鲁棒性,从而使得移动机器人能够在较大的距离范围内快速、可靠地识别室内各种典型障碍物.     

Immune response to autologous and heterologous Helicobacter pylori antigens in humans

Infection of humans with Helicobacter pylori results in the development of chronic gastritis and plays an important role in gastric ulcer pathogenesis. Despite the infiltration of the mucosa with specific immunocompetent cells and production of specific antibodies, the infection usually persists for life. This study was performed to investigate if immunologic mechanisms exist which could contribute to the inability of the host to terminate the infection. Therefore, we compared the in vitro immunoreactivity of peripheral blood mononuclear cells (PBMC) from H. pylori-infected patients after stimulation with sonicated H. pylori bacteria from the stomach of the patient (autologous bacterial strain) with stimulation by bacteria from other patients (heterologous bacteria). We measured cell proliferation, expression of T cell activation markers CD25, HLA-DR, and CD71, as well as production ofinterleukin-10 (IL-10), an inhibitory cytokine. We found that the proliferative response of PBMC was significantly lower after autologous than after heterologous stimulation. Furthermore, secretion of IL-10 in the culture supernatants was significantly higher when PBMC were incubated with autologous than with heterologous H. pylori antigens. No significant differences between autologous or heterologous stimulation were observed in the increased expression of T cell activation markers. These data indicate that systemic immunologic response to H. pylori are strain-dependent. For further studies of the immune responses towards H. pylori, the use of an autologous stimulatory system seems necessary.     

GP30 of the mycobacteriophage CASbig impairs mycobacterial adaptation during acidic stress and in macrophages

The rapid emergence of multidrug-resistant and extensively drug-resistant Tuberculosis retrieved intense interest in phage-based therapy. This old approach, which was abandoned in the west in the 1940s but is generating renewed interest, has stimulated fresh research on mycobacteriophages and their lytic efficiency against their hosts. GP30 is a novel protein of the mycobacteriophage CASbig with undiscovered function. In this study, we analyzed the role of CASbig gp30 in the host Mycobacterium smegmatis. Overexpression of gp30 in the host led to reduced growth in acidic medium and attenuated the intracellular survival rate of M. smegmatis inside the THP-1 macrophages, which may be linked to the altered lipid profile of the recombinant bacterial cell wall. In a word, this study suggested that gp30, a novel gene from a mycobacteriophage, modulated lipid composition and content to hamper the survivability of bacteria under stress conditions.     

Immobilisation of living bacteria for AFM imaging under physiological conditions

Atomic force microscopy (AFM) holds great potential for studying the nanoscale surface structures of living cells, and to measure their interactions with abiotic surfaces, other cells, or specific biomolecules. However, the application of AFM in microbiology is challenging due to the difficulty of immobilising bacterial cells to a flat surface without changing the cell surface properties or cell viability. We have performed an extensive and thorough study of how to functionalise surfaces in order to immobilise living bacteria for AFM studies in liquid environments. Our aim was to develop a scheme which allows bacterial cells to be immobilised to a flat surface with sufficient strength to avoid detachment during the AFM scanning, and without affecting cell surface chemistry, structure, and viability. We compare and evaluate published methods, and present a new, reproducible, and generally applicable scheme for immobilising bacteria cells for an AFM imaging.     

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM – SEM)

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.     

求解作业车间调度的变邻域细菌觅食优化算法

针对最小化最大完工时间的作业车间调度问题,提出一种基于变邻域趋化操作的细菌觅食优化算法。邻域搜索是一类改进型局部搜索算法,在每一步迭代过程中通过搜索当前解的邻域得到一个改进的解,利用邻域搜索可大大提高局部最优解的精确度。本算法采用基于操作的编码,使得细菌觅食优化算法适用于作业车间调度求解;将3种不同的邻域结构引入趋化操作中,以便扩大可行解的搜索空间,细菌个体按照自适应学习策略根据邻域的各自贡献率选择搜索方式,减少陷入局部极小的机会;同时使用自适应步长更新各邻域内趋化操作的位置,根据适应度值动态调整搜索精度,避免早熟收敛。典型算例试验表明,该算法具有一定的鲁棒性,并有效地提高了搜索精度和收敛性。     

The role of the plant cytoskeleton in the interaction between legumes and rhizobia

The study of the symbiotic interaction between rhizobia and legumes represents a major theme in plant biology. This interaction results in the formation of nodules, root organs in which the bacteria reduce atmospheric nitrogen into ammonia, which can subsequently be utilized by the plant. The execution of the different developmental stages observed during nodule ontogenesis involves many cellular processes with significant roles for the plant cytoskeleton. A challenging question in cell biology is how the cytoskeleton organizes itself into the dynamic arrays required for cell differentiation and functioning. Nodulation is, particularly, well qualified as an experimental system for cytoskeleton research because an early essential step of the plant/microbe interaction takes place in surface-exposed root hairs, well suited for cell biological in vivo experimentation. Moreover, the changes in the organization of the cytoskeleton can be elicited by a well-defined molecule, the Nod factor, or by bacterial inoculation, thus providing the researcher with the possibility of controlling the cytoskeletal changes in target cells. In addition, the well-known cytology of the symbiotic interaction facilitates the correlation between the changes in the organization of the plant cytoskeleton with both histological and cellular changes. In this review, the current knowledge on the role of the plant cytoskeleton during nodulation is summarized, with emphasis on the interaction between Medicago truncatula and Sinorhizobium meliloti .     

The effect of exit beam phase aberrations on parallel beam coherent x-ray reconstructions

Diffraction artifacts from imperfect x-ray windows near the sample are an important consideration in the design of coherent x-ray diffraction measurements. In this study, we used simulated and experimental diffraction patterns in two and three dimensions to explore the effect of phase imperfections in a beryllium window (such as a void or inclusion) on the convergence behavior of phasing algorithms and on the ultimate reconstruction. A predictive relationship between beam wavelength, sample size, and window position was derived to explain the dependence of reconstruction quality on beryllium defect size. Defects corresponding to this prediction cause the most damage to the sample exit wave and induce signature error oscillations during phasing that can be used as a fingerprint of experimental x-ray window artifacts. The relationship between x-ray window imperfection size and coherent x-ray diffractive imaging reconstruction quality explored in this work can play an important role in designing high-resolution in situ coherent imaging instrumentation and will help interpret the phasing behavior of coherent diffraction measured in these in situ environments.     

AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces

Immobilization of particulates, especially biomolecules and cells, onto surfaces is critical for imaging with the atomic force microscope (AFM). In this paper, gelatin coated mica surfaces are shown to be suitable for immobilizing and imaging both gram positive, Staphylococcus aureus, and gram negative, Escherichia coli, bacteria in both air and liquid environments. Gelatin coated surfaces are shown to be superior to poly-L-lysine coated surfaces that are commonly used for the immobilization of cells. This cell immobilization technique is being developed primarily for live cell imaging of Rhodopseudomonas palustris. The genome of R. palustris has been sequenced and the organism is the target of intensive studies aimed at understanding genome function. Images of R. palustris grown both aerobically and anaerobically in liquid media are presented. Images in liquid media show the bacteria is rod shaped and smooth while images in air show marked irregularity and folding of the surface. Significant differences in the vertical dimension are also apparent with the height of the bacteria in liquid being substantially greater than images taken in air. In air immobilized bacterial flagella are clearly seen while in liquid this structure is not visible. Additionally, significant morphological differences are observed that depend on the method of bacterial growth.