Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.Members of the genus Bacillus are rod-shaped bacteria that exhibit catalase activity and can be characterized as endospore-forming obligate or facultative aerobes. The genus Bacillus contains two important groups of bacteria named after B. subtilis and B. cereus. The best-characterized member of the former group is B. subtilis, a renowned model organism for genetic research. Other group members, like B. pumilis, B. licheniformis, B. atrophaeus, and B. amyloliquefaciens, exhibit a high degree of phenotypic similarity and are thus not easily distinguishable (15).The B. cereus group comprises a number of closely related bacteria, some of which interfere with human health. Bacteria classified as B. cereus are occasionally associated with food poisoning (16, 28), while B. thuringiensis is primarily an insect pathogen because of its ability to produce toxins that have been widely used for the biocontrol of insect pests (28, 30). A third member of the B. cereus group, B. anthracis, is the causative agent of anthrax and is highly relevant to human and animal health. Other members of the B. cereus group are B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 15).B. anthracis is a possible agent in biological warfare and bioterrorism. Its applicability as a biological warfare agent was made apparent by an accidental release from a Soviet military facility in Sverdlovsk (1, 10). Also, the well-publicized mailing of B. anthracis spores in the United States, which caused 18 confirmed cases of cutaneous and inhalational anthrax and an additional 4 suspected cases of cutaneous anthrax (3, 22), demonstrated that B. anthracis may become a threat from terrorist groups (10).Rapid detection of B. anthracis may be challenging because of its great genetic similarity to other species of the B. cereus group (10) and the difficulties of phenotypic differentiation of B. cereus group members (15). There is some controversy in the literature regarding the taxonomy of the B. cereus group. Indeed, some authors state that B. anthracis, B. cereus, and B. thuringiensis are one species with various virulence plasmids for the toxin pXO1 and the capsule pXO2 of B. anthracis and the insecticidal toxin of B. thuringiensis (10, 19). Other authors do not support this opinion and suggest the presence of even more species within the group (21).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) intact-cell mass spectrometry (ICMS) has been suggested as a rapid, objective, and reliable technique for bacterial identification (8, 13, 23, 25, 38). As a proteomic technique, ICMS of whole bacterial cells, or cell lysates, relies on the reproducible detection of microbial protein patterns and thus delivers information complementary to genotypic or phenotypic test methods. With the pattern-matching approach, microbial identification is achieved by comparing experimental mass spectra with a collection of mass spectra of known organisms. This requires the compilation of large databases of bacterial reference spectra but has the advantage that an extensive knowledge of biomarker identities is not required. Another advantage of the pattern-matching approach is that genus- and species-specific procedures or consumables are not required, i.e., the same methodology can in principle be applied to all kinds of microorganisms (multiplex advantage).It is thus believed that ICMS offers the possibility to systematically investigate the diversity of bacterial subproteomes, complementing existing methodologies of bacterial characterization. This potential and the need for a rapid, objective, and reliable microbial identification technique that does not rely on nucleic acid detection and the availability of an MS-compatible inactivation protocol for highly pathogenic biosafety level 3 microorganisms and bacterial endospores (26) prompted us to systematically study the MALDI-TOF MS profiles of Bacillus strains and to establish a database of bacterial mass spectra. In the present work, we describe strategies of spectral analysis that allow the identification and validation of group- and species-specific sets of biomarkers. Using unsupervised hierarchical cluster analysis (UHCA) and supervised artificial neural network (ANN) analysis, we also demonstrate how microbial spectra can be employed to establish an MS-based methodology for rapid, objective, and reliable identification of the target species, B. anthracis.     

The N Terminus of Connexin37 Contains an ��-Helix That Is Required for Channel Function

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly α-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the α-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.Gap junction channels allow intercellular passage of ions and small molecules up to 1000 Da. They are oligomeric assemblies of members of a family of related proteins called connexins (CX)² (reviewed in Ref. 1). Six connexin monomers assemble to form a hemichannel or connexon (Fig. 1, top panel), which, in turn, forms a complete gap junction channel by docking with a hemichannel from an adjacent cell. Based on sequence similarities, connexins have been separated into subfamilies designated by Greek characters (2, 3). The majority of connexins are members of the α- and β-subfamilies. Connexin polypeptides span the plasma membrane four times and have three cytoplasmic regions: the N terminus (NT), a cytoplasmic loop between the second and third transmembrane domains, and the C terminus (Fig. 1, middle panel). Structural studies of gap junctions have revealed that each hemichannel contains a ring of 24 transmembrane spanning helices (4, 5). Most topological models suggest that the NT of α-subfamily connexins contains 23 amino acids (illustrated for connexin37, CX37, in Fig. 1, bottom panel) and that of β-subfamily connexins contains 22 amino acids.Open in a separate windowFIGURE 1.Diagrams depicting the relationships between a gap junction hemichannel (top), the connexin polypeptide (middle), and the amino acid sequence of the CX37 N-terminal domain (bottom). Thick vertical lines represent the boundaries of the plasma membrane; the intracellular and extracellular spaces are indicated. The transmembrane (M1–M4), extracellular (E1 and E2), and cytoplasmic (NT, N terminus; CL, cytoplasmic loop; and CT, C terminus) domains within a connexin are indicated.The importance of the connexin NT has been emphasized by the identification of a number of connexin mutants that cause amino acid substitutions within this region and are linked to diseases including sensorineural deafness (CX26, CX30, and CX31), Charcot-Marie-Tooth disease (CX32), oculodentodigital dysplasia (CX43), and congenital cataracts (CX46 and CX50). Among the disease-linked mutants that have been studied, some show impaired protein trafficking to the cell surface, whereas others traffic properly, but show loss or alterations of channel function (6–16). Heterologous expression of site-directed mutants and chimeric connexins has demonstrated the influence of NT amino acids upon channel properties, including transjunctional voltage (V_j)-dependent gating, unitary conductance, permeability, and sensitivity to regulation by polyamines (17–22). Lagree et al. (23) have provided evidence that the NT influences the compatibility of connexin hetero-oligomerization.The structure of the NT domain of a β-group connexin, Cx26, has been investigated through circular dichroism (CD) and nuclear magnetic resonance (NMR) of a synthetic peptide corresponding to part of the predicted CX26NT (24, 25). Based on their data, Purnick et al. (24) proposed a model for the NT of CX26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel. Oshima et al. (5) have published structural studies of a “permeability” mutant (M34A) of CX26 (26) showing a density within the pore of the channel that they suggested might represent a bundle of N termini acting as a “plug” to close the channel.We have been studying CX37, an α-group connexin that is expressed in endothelial cells (27), which may be important for development of atherosclerotic disease (28) and that can form large conductance channels and hemichannels (27, 29). We have shown that as much as half the length of the CX37NT can be deleted without affecting formation of gap junction plaques, but a full-length N terminus is required for hemichannel gating and intercellular communication (30). These observations suggested that the CX37NT may have a structure that is required for function. Therefore, the present experiments were designed to determine the structure of the NT of CX37 and the importance of that structure for protein localization and formation of functional channels and hemichannels. Differences between our data and those previously reported in studies of CX26 suggest that the structure of the NT in α-group connexins may differ from that in β-group connexins.     

Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis

Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.     

Accounting for Redundancy when Integrating Gene Interaction Databases

During the last years gene interaction networks are increasingly being used for the assessment and interpretation of biological measurements. Knowledge of the interaction partners of an unknown protein allows scientists to understand the complex relationships between genetic products, helps to reveal unknown biological functions and pathways, and get a more detailed picture of an organism''s complexity. Being able to measure all protein interactions under all relevant conditions is virtually impossible. Hence, computational methods integrating different datasets for predicting gene interactions are needed. However, when integrating different sources one has to account for the fact that some parts of the information may be redundant, which may lead to an overestimation of the true likelihood of an interaction. Our method integrates information derived from three different databases (Bioverse, HiMAP and STRING) for predicting human gene interactions. A Bayesian approach was implemented in order to integrate the different data sources on a common quantitative scale. An important assumption of the Bayesian integration is independence of the input data (features). Our study shows that the conditional dependency cannot be ignored when combining gene interaction databases that rely on partially overlapping input data. In addition, we show how the correlation structure between the databases can be detected and we propose a linear model to correct for this bias. Benchmarking the results against two independent reference data sets shows that the integrated model outperforms the individual datasets. Our method provides an intuitive strategy for weighting the different features while accounting for their conditional dependencies.     

Binding of alpha-synuclein affects the lipid packing in bilayers of small vesicles

The intracellular deposition of fibrillar aggregates of alpha-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, alpha-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that alpha-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that alpha-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of alpha-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that alpha-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.     

Seed dormancy release and germination characteristics of <i>Corispermum lehmannianum</i> Bunge,an endemic species in the Gurbantunggut desert of China

Seed dormancy release and germination of Corispermum lehmannianum Bunge were tested using various treatments: temperature, cold stratification, gibberelins (GA3), dry storage and sand burial. Results showed that temperature and light did not affect the germination of fresh seeds, cold stratification and GA3 could improve seed germination, whereas dry storage and sand burial did not. The germination percentage was highest at 35/20 °C after the cold stratification and GA3 treatments. Corispermum lehmannianum seeds were classified as non-deep, Type-2, physiological dormancy (PD), whose seed dormancy could be released by cold stratification and GA3.     

Upregulation and phosphorylation of HspB1/Hsp25 and HspB5/αB-crystallin after transient middle cerebral artery occlusion in rats

Ischemic stroke leads to cellular dysfunction, cell death, and devastating clinical outcomes. The cells of the brain react to such a cellular stress by a stress response with an upregulation of heat shock proteins resulting in activation of endogenous neuroprotective capacities. Several members of the family of small heat shock proteins (HspBs) have been shown to be neuroprotective. However, yet no systematic study examined all HspBs during cerebral ischemia. Here, we performed a comprehensive comparative study comprising all HspBs in an animal model of stroke, i.e., 1 h transient middle cerebral artery occlusion followed by 23 h of reperfusion. On the mRNA level out of the 11 HspBs investigated, HspB1/Hsp25, HspB3, HspB4/αA-crystallin, HspB5/αB-crystallin, HspB7/cvHsp, and HspB8/Hsp22 were significantly upregulated in the peri-infarct region of the cerebral cortex of infarcted hemispheres. HspB1 and HspB5 reached the highest mRNA levels and were also upregulated at the protein level, suggesting that these HspBs might be functionally most relevant. Interestingly, in the infarcted cortex, both HspB1 and HspB5 were mainly allocated to neurons and to a lesser extent to glial cells. Additionally, both proteins were found to be phosphorylated in response to ischemia. Our data suggest that among all HspBs, HspB1 and HspB5 might be most important in the neuronal stress response to ischemia/reperfusion injury in the brain and might be involved in neuroprotection.