口腔微生物与全身健康研究进展

口腔微生物是定植于人体口腔的微生物集合。众多研究证实,口腔微生物与多种口腔感染性疾病及系统性疾病紧密相关。随着“人类微生物组计划”及其他微生物宏基因组学相关项目的开展,人们对口腔微生物群落的认识不断深入。本文基于最新研究进展,就口腔微生物的组成、演替特点、与口腔和全身系统性疾病的关系及与肠道微生物的交互作用进行综述。     

A novel approach for extracting viscoelastic parameters of living cells through combination of inverse finite element simulation and Atomic Force Microscopy

Dynamic mechanical behaviour of living cells has been described by viscoelasticity. However, quantitation of the viscoelastic parameters for living cells is far from sophisticated. In this paper, combining inverse finite element (FE) simulation with Atomic Force Microscope characterization, we attempt to develop a new method to evaluate and acquire trustworthy viscoelastic index of living cells. First, influence of the experiment parameters on stress relaxation process is assessed using FE simulation. As suggested by the simulations, cell height has negligible impact on shape of the force–time curve, i.e. the characteristic relaxation time; and the effect originates from substrate can be totally eliminated when stiff substrate (Young’s modulus larger than 3 GPa) is used. Then, so as to develop an effective optimization strategy for the inverse FE simulation, the parameters sensitivity evaluation is performed for Young’s modulus, Poisson’s ratio, and characteristic relaxation time. With the experiment data obtained through typical stress relaxation measurement, viscoelastic parameters are extracted through the inverse FE simulation by comparing the simulation results and experimental measurements. Finally, reliability of the acquired mechanical parameters is verified with different load experiments performed on the same cell.     

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.     

Liquid chromatography-electrospray ionisation mass spectrometry for the determination of nine selected benzodiazepines in human plasma and oral fluid

A new simple and rapid liquid chromatographic-mass spectrometric technique was designed for the determination of nine benzodiazepines in plasma and oral fluid. Benzodiazepines were extracted from alkalinised spiked and clinical plasma and oral fluid samples using a single step, liquid-liquid extraction procedure with diethyl ether. The chromatographic separation was performed with a Xterra RP18, 5 microm (150 x 2.1 mm i.d.) reversed-phase column using deuterated analogues of the analytes as internal standard. The recovery ranged from 70.3 to 86.9% for plasma and 63.9 to 77.2% for oral fluid. The limits of detection ranged from 0.5 to 1 ng/ml in plasma and 0.1 to 0.2 ng/ml for oral fluid. The method was validated for all the compounds, including linearity and the main precision parameters. The procedure, showed to be sensitive and specific, was applied to real plasma and oral fluid samples. The method is especially useful to analyse saliva samples from drivers undergoing roadside drug controls.     

Comparative analysis of cell surface proteins in chronic and acute leukemia cell lines

This study was designed to identify the cell surface protein markers that can differentiate between chronic myeloid leukemia (CML) and acute promyelocytic leukemia cells (APL). The differentially expressed plasma membrane proteins were analyzed between CML cell line (K562) and APL cell line (NB4) using the comparative proteomic approach. The cell membrane proteins were enriched by labeling with a membrane-impermeable biotinylation reagent, sulfo-NHS-SS-Biotin, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). By comparative proteomic analysis of K562 and NB4 cells, we identified 25 membrane and 14 membrane-associated proteins. The result of LC-MS/MS combined with chemical tagging method was validated by confirming the expression and localization of one of the differentially expressed plasma membrane proteins, CD43, by FACS and confocal microscopy. Our results indicate that CD43 could be a potential candidate for differentiating CML from APL.     

Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry

To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)₂, blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.     

NMR resonance assignments for sparsely <Superscript>15</Superscript>N labeled proteins

For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform ¹³C, ¹⁵N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected ¹⁵N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform ¹⁵N, ¹³C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach applicable to a single sample prepared with sparse ¹⁵N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein, Galectin-3. Limitations and future applications are discussed.     

Efficient removal of detergents from proteins and peptides in a spin column format

Detergents are commonly used in protein–chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1–5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography–tandem MS (LC–MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)–MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15 min), efficient detergent removal, and high recovery of proteins and peptides.     

Identification of proteins found to be significantly altered when comparing the serum proteome from Multiple Myeloma patients with varying degrees of bone disease

Background

Bone destruction is a feature of multiple myeloma, characterised by osteolytic bone destruction due to increased osteoclast activity and suppressed or absent osteoblast activity. Almost all multiple myeloma patients develop osteolytic bone lesions associated with severe and debilitating bone pain, pathologic fractures, hypercalcemia, and spinal cord compression, as well as increased mortality. Biomarkers of bone remodelling are used to identify disease characteristics that can help select the optimal management of patients. However, more accurate biomarkers are needed to effectively mirror the dynamics of bone disease activity.

Results

A label-free mass spectrometry-based strategy was employed for discovery phase analysis of fractionated patient serum samples associated with no or high bone disease. A number of proteins were identified which were statistically significantly correlated with bone disease, including enzymes, extracellular matrix glycoproteins, and components of the complement system.

Conclusions

Enzyme-linked immunosorbent assay of complement C4 and serum paraoxonase/arylesterase 1 indicated that these proteins were associated with high bone disease in a larger independent cohort of patient samples. These biomolecules may therefore be clinically useful in assessing the extent of bone disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-904) contains supplementary material, which is available to authorized users.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

Proteomic detection of proteins involved in perchlorate and chlorate metabolism

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.     

Peripheral blood values as predictors of autoimmune status in oral cavity squamous cell carcinoma

BackgroundRecent literature has highlighted the role of the host in prognosis in oral squamous cell carcinoma (OSCC). Autoimmune (AI) disease represents a macroscopic depiction of host status. The goal of this study was to predict an AI “status” and to analyze the utility of this “status” as a prognostic indicator in OSCC.MethodsFrom a departmental database of OSCC patients (n = 1377), 125 patients with an AI disorder were identified. PBL values were obtained and standardized for analysis. A LASSO regression model was used to determine the best predictors of AI status and an AI score was developed. The score was then analyzed across various survival endpoints.ResultsWhen AI score was divided into a binary variable, patients in the highest quartile had a significantly worse overall survival (OS), local recurrence-free (LRFP) and distant recurrence-free probability (DRFP). Survival curves showed significant differences for OS, DSS, LRFP, and DRFP.ConclusionsAI diseases are immune dysregulations that could play a role in prognosis. Therefore, development of an AI score is necessary to depict host status in a ubiquitous manner. AI score as a binary variable may be more utilitarian in a clinical setting, compared to the continuous score. This novel tool needs validation and integration into more tumor and host characteristics. This investigation showed utility of such a score, similar to PBL data in OSCC prognosis. Future studies should incorporate other relevant variables known to affect outcome and implement a more comprehensive predictive model.     

Speciation of coagulase negative staphylococci, isolated from indoor air, using SDS PAGE gel bands of expressed proteins followed by MALDI TOF MS and MALDI TOF-TOF MS-MS analysis of tryptic peptides

A group of coagulase negative staphylococcal strains isolated from indoor air of occupied school rooms were the subject of this study. Conventional MALDI TOF MS profiling of cellular extracts and physiological tests (including API STAPH) provided incomplete identification of the set of strains. After separation of a 100 kDa band using 1D gel electrophoresis, profiling of peptides (released with tryptic digestion) using MALDI TOF MS allowed improved bacterial speciation in addition to determination of the identity of the protein of origin (aconitate hydratase). This was performed by Mascot search, empirical observation and computer-generated cross-correlation analysis of environmental isolates versus reference strains. The species studied included some with sequenced genomes and others with un-sequenced genomes. Peptide sequences were confirmed to originate from aconitate hydratase using MALDI TOF-TOF MS-MS analysis of a diverse set of m/z values representing variable and conserved sequences. The methodological approach described here might have widespread application in speciation of environmental isolates of diverse origin and in identification of their expressed proteins.     

Proteome analysis of mouse model systems: A tool to model human disease and for the investigation of tissue-specific biology

The molecular dissections of the mechanistic pathways involved in human disease have always relied on the use of model organisms. Among the higher mammalian organisms, the laboratory mouse (Mus musculus) is the most widely used model. A large number of commercially-available, inbred strains are available to the community, including an ever growing collection of transgenic, knock-out, and disease models. Coupled to availability is the fact that animal colonies can be kept under standardized housing condition at most major universities and research institutes, with relative ease and cost efficiency (compared to larger vertebrates). As such, mouse models to study human biology and disease remains extremely attractive. In the current review we will provide an historic overview of the use of mouse models in proteome research with a focus on general tissue and organelle biology, comparative proteomics of human and mouse and the use of mouse models to study cardiac disease.     

Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics

Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match. This review discusses the basic principles of spectral library building and searching, describes its advantages and limitations, and provides a primer for researchers interested in adopting this new approach in their data analysis. It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics.     

Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry

An oral sorbent AST-120 composed of spherical porous carbon particles has superior adsorption ability for certain small-molecular-weight organic compounds known to accumulate in patients with chronic renal failure (CRF). A metabolomic approach was applied to search for uremic toxins as possible indicators of the effect of AST-120. Serum metabolites in normal and CRF rats before and after administration of AST-120 for 3 days were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis. Further, serum and urine levels of the indicators were quantified by selected reaction monitoring of LC/ESI-MS/MS. Indoxyl sulfate was the first principal serum metabolite, which could differentiate CRF from both normal and AST-120-administered CRF rats, followed by hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate. CRF rats showed increased serum levels of indoxyl sulfate, hippuric acid, phenyl sulfate, 4-ethylphenyl sulfate and p-cresyl sulfate. Administration of AST-120 for 3 days to the CRF rats reduced the serum and urine levels of these metabolites. In conclusion, indoxyl sulfate is the best indicator of the effect of AST-120 in CRF rats. Hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate are suggested as the additional indicators. 4-Ethylphenyl sulfate is a newly identified uremic substance.     

Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption

A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.