Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

Investigation of receptor interacting protein (RIP3)-dependent protein phosphorylation by quantitative phosphoproteomics.

Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3(+/+)) and RIP3 knockout (RIP3(-/-)) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3(+/+) and RIP3(-/-) mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3(+/+) and RIP3(-/-) mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3(+/+) macrophages, 121 were detected exclusively from RIP3(+/+) MEFs, 286 phosphopeptides were induced more in RIP3(+/+) macrophages than in RIP3(-/-) macrophages and 26 phosphopeptides had higher induction in RIP3(+/+) MEFs than in RIP3(-/-) cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists.

Quartet metabolite reference materials for inter-laboratory proficiency test and data integration of metabolomics profiling.

BACKGROUND: Various laboratory-developed metabolomic methods lead to big challenges in inter-laboratory comparability and effective integration of diverse datasets. RESULTS: As part of the Quartet Project, we establish a publicly available suite of four metabolite reference materials derived from B lymphoblastoid cell lines from a family of parents and monozygotic twin daughters. We generate comprehensive LC-MS-based metabolomic data from the Quartet reference materials using targeted and untargeted strategies in different laboratories. The Quartet multi-sample-based signal-to-noise ratio enables objective assessment of the reliability of intra-batch and cross-batch metabolomics profiling in detecting intrinsic biological differences among the four groups of samples. Significant variations in the reliability of the metabolomics profiling are identified across laboratories. Importantly, ratio-based metabolomics profiling, by scaling the absolute values of a study sample relative to those of a common reference sample, enables cross-laboratory quantitative data integration. Thus, we construct the ratio-based high-confidence reference datasets between two reference samples, providing "ground truth" for inter-laboratory accuracy assessment, which enables objective evaluation of quantitative metabolomics profiling using various instruments and protocols. CONCLUSIONS: Our study provides the community with rich resources and best practices for inter-laboratory proficiency tests and data integration, ensuring reliability of large-scale and longitudinal metabolomic studies.

Multi-stage metabolomics and genetic analyses identified metabolite biomarkers of metabolic syndrome and their genetic determinants.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of multiple cardiometabolic risk factors that increase the risk of type 2 diabetes and cardiovascular diseases. Identifying novel biomarkers of MetS and their genetic associations could provide insights into the mechanisms of cardiometabolic diseases. METHODS: Potential MetS-associated metabolites were screened and internally validated by untargeted metabolomics analyses among 693 patients with MetS and 705 controls. External validation was conducted using two well-established targeted metabolomic methods among 149 patients with MetS and 253 controls. The genetic associations of metabolites were determined by linear regression using our previous genome-wide SNP data. Causal relationships were assessed using a one-sample Mendelian Randomization (MR) approach. FINDINGS: Nine metabolites were ultimately found to be associated with MetS or its components. Five metabolites, including LysoPC(14:0), LysoPC(15:0), propionyl carnitine, phenylalanine, and docosapentaenoic acid (DPA) were selected to construct a metabolite risk score (MRS), which was found to have a dose-response relationship with MetS and metabolic abnormalities. Moreover, MRS displayed a good ability to differentiate MetS and metabolic abnormalities. Three SNPs (rs11635491, rs7067822, and rs1952458) were associated with LysoPC(15:0). Two SNPs, rs1952458 and rs11635491 were found to be marginally correlated with several MetS components. MR analyses showed that a higher LysoPC(15:0) level was causally associated with the risk of overweight/obesity, dyslipidaemia, high uric acid, high insulin and high HOMA-IR. INTERPRETATION: We identified five metabolite biomarkers of MetS and three SNPs associated with LysoPC(15:0). MR analyses revealed that abnormal LysoPC metabolism may be causally linked the metabolic risk. FUNDING: This work was supported by grants from the National Key Research and Development Program of China (2017YFC0907004).

Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis.

Drought is one of the most severe limitations to plant growth and productivity. Resurrection plants have evolved a unique capability to tolerate desiccation in vegetative tissues. Fern-ally Selaginella tamariscina (Beauv.) is one of the most primitive vascular resurrection plants, which can survive a desiccated state and recover when water becomes available. To better understand the mechanism of desiccation tolerance, we have applied physiological and proteomic analysis. Samples of S. tamariscina were water-deprived for up to seven days followed by 12 h of rewatering. Our results showed that endogenous abscisic acid (ABA) increased to regulate dehydration-responsive genes/proteins and physiological processes. In the course of dehydration, the contents of osmolytes represented by soluble sugars and proline were increased to maintain cell structure integrity. The activities of four antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR)) also increased. In contrast, both the rate of photosynthesis and the chlorophyll content decreased, and plasma membrane integrity was lost. We identified 138 desiccation-responsive two-dimensional electrophoresis (2-DE) spots, representing 103 unique proteins. Hierarchical clustering analysis revealed that 83% of the proteins were down-regulated upon dehydration. They were mainly involved in photosynthesis, carbohydrate and energy metabolism, stress and defense, protein metabolism, signaling, membrane/transport, cell structure, and cell division. The dynamic expression changes of the desiccation-responsive proteins provide strong evidence that cell structure modification, photosynthesis reduction, antioxidant system activation, and protein post-transcriptional/translational modifications are essential to the poikilochlorophyllous fern-ally S. tamariscina in response to dehydration. In addition, our comparative analysis of dehydration-responsive proteins in vegetative tissues from 19 desiccation tolerant and nontolerant plant species suggests that resurrection S. tamariscina has developed a specific desiccation tolerant mechanism. To our knowledge, this study constitutes the first detailed investigation of the protein complement in fern/fern-allies.

Combination of improved (18)O incorporation and multiple reaction monitoring: a universal strategy for absolute quantitative verification of serum candidate biomarkers of liver cancer.

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS), which is an alternative to immunoassay methods such as ELISA and Western blotting, has been used to alleviate the bottlenecks of high-throughput verification of biomarker candidates recently. However, the inconvenience and high isotope consumption required to obtain stably labeled peptide impedes the broad application of this method. In our study, the (18)O-labeling method was introduced to generate stable isotope-labeled peptides instead of the Fmoc chemical synthesis and Qconcat recombinant protein synthesis methods. To make (18)O-labeling suitable for absolute quantification, we have added the following procedures: (1) RapiGest SF and microwave heating were added to increase the labeling efficiency; (2) trypsin was deactivated completely by chemical modification using tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA) to prevent back-exchange of (18)O to (16)O, and (3) MRM parameters were optimized to maximize specificity and better distinguish between (18)O-labeled and unlabeled peptides. As a result, the (18)O-labeled peptides can be prepared in less than 1 h with satisfactory efficiency (>97%) and remained stable for 1 week, compared to traditional protocols that require 5 h for labeling with poor stability. Excellent separation of (18)O-labeled and unlabeled peptides was achieved by the MRM-MS spectrum. Finally, through the combined improvement in (18)O-labeling with multiple reaction monitoring, an absolute quantification strategy was developed to quantitatively verify hepatocellular carcinoma-related biomarker candidates, namely, vitronectin and clusterin, in undepleted serum samples. Sample preparation and capillary-HPLC analysis were optimized for high-throughput applications. The reliability of this strategy was further evaluated by method validation, with accuracy (%RE) and precision (%RSD) of less than 20% and good linearity (r(2) > 0.99), and clinical validation, which were consistent with previously reported results. In summary, our strategy can promote broader application of SID-MRM-MS for biomarkers from discovery to verification regarding the significant advantages of the convenient and flexible generation of internal standards, the reduction in the sample labeling steps, and the simple transition.

Identification of tetranectin as a potential biomarker for metastatic oral cancer.

Lymph node involvement is the most important predictor of survival rates in patients with oral squamous cell carcinoma (OSCC). A biomarker that can indicate lymph node metastasis would be valuable to classify patients with OSCC for optimal treatment. In this study, we have performed a serum proteomic analysis of OSCC using 2-D gel electrophoresis and liquid chromatography/tandem mass spectrometry. One of the down-regulated proteins in OSCC was identified as tetranectin, which is a protein encoded by the CLEC3B gene (C-type lectin domain family 3, member B). We further tested the protein level in serum and saliva from patients with lymph-node metastatic and primary OSCC. Tetranectin was found significantly under-expressed in both serum and saliva of metastatic OSCC compared to primary OSCC. Our results suggest that serum or saliva tetranectin may serve as a potential biomarker for metastatic OSCC. Other candidate serum biomarkers for OSCC included superoxide dismutase, ficolin 2, CD-5 antigen-like protein, RalA binding protein 1, plasma retinol-binding protein and transthyretin. Their clinical utility for OSCC detection remains to be further tested in cancer patients.

Glycoprofiling of the Human Salivary Proteome.

Glycosylation is important for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins. This work combines two-dimensional polyacrylamide gel electrophoresis (2-DE) and lectin blotting to map the salivary glycome, and mass spectrometry to identity the proteins that are associated with the glycome map. A panel of 15 lectins that recognize six sugar-specific categories was used to visualize the type and extent of glycosylation in saliva from two healthy male individuals. Lectin blots were compared to 2-D gels stained either with Sypro Ruby (protein stain) or Pro-Q Emerald 488 (glycoprotein stain). Each lectin shows a distinct pattern, even those belonging to the same sugar-specific category. In addition, the glycosylation profiles generated from the lectin blots show that most of the salivary proteins are glycosylated and that the pattern is more widespread than is demonstrated by the glycoprotein stained gel. Finally, the co-reactivity between two lectins was measured to determine the glycan structures that are most and least often associated with one another along with the population variation of the lectin reactivity for 66 individuals.

Identification and characterization of the chromium (VI) responding protein from a newly isolated Ochrobactrum anthropi CTS-325.

A Gram-negative, chromium(VI) tolerant and reductive strain CTS-325, isolated from a Chinese chromate plant, was identified as Ochrobactrum anthropi based on its biochemical properties and 16S rDNA sequence analysis. It was able to tolerate up to 10 mmol/L Cr(VI) and completely reduce 1 mmol/L Cr(VI) to Cr(III) within 48 h. When the strain CTS-325 was induced with Cr(VI), a protein increased significantly in the whole cell proteins. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that this protein was a superoxide dismutase (SOD) homology. The measured superoxide dismutase activity was 2694 U/mg after three steps of purification. The SOD catalyzes the dismutation of the superoxide anion (O2*-) into hydrogen peroxide and molecular oxygen. This protein is considered to be one of the most important anti-oxidative enzymes for O. anthropi as it allows the bacterium to survive high oxygen stress environments, such as the environment produced during the reduction process of Cr(VI).

Mass spectrometry of protein-ligand complexes: enhanced gas-phase stability of ribonuclease-nucleotide complexes.

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.

Proteomic analysis to characterize differential mouse strain sensitivity to cadmium-induced forelimb teratogenesis.

BACKGROUND: Cadmium ion (Cd2+) is a ubiquitous environmental contaminant, and it is a potent teratogen in mice. An intraperitoneal dose of 4 mg/kg of CdCl2 at gestational day 9 causes forelimb ectrodactyly in the C57BL/6N mouse strain, but the SWV/Fnn strain is resistant. The objective of this study was to identify differentially displayed proteins in two target tissues for cadmium teratogenesis, and to derive hypotheses regarding the mechanisms involved in the murine strain difference in Cd-induced forelimb ectrodactyly. METHODS: The global proteomics strategy used two-dimensional polyacrylamide gel electrophoresis for protein separation, and MALDI-TOF-MS and LC-MS/MS for protein identification, to compare and identify proteins in forelimb buds and yolk sacs from the two mouse strains following Cd administration. RESULTS: More than 1,000 protein spots were detected by two-dimensional polyacrylamide gel electrophoresis in day 10.0 mouse forelimb buds and yolk sacs. Thirty-eight proteins had identifiable differences in abundance levels in Cd-treated forelimb buds between the two strains. Of those 38 proteins, 14 could be associated with the unfolded protein response process and seven are associated with actin polymerization. The proteins that were found to be differentially abundant between the strains in yolk sacs that were exposed to CdCl2 were predominantly different than the proteins detected differentially in the limb buds of the two strains with an overlap of approximately 20%. CONCLUSIONS: These patterns of differentially displayed proteins rationalize a hypothesis that the differential murine strain response to cadmium-induced forelimb ectrodactyly is due to differences in their pathways for the unfolded protein response and/or actin polymerization.

Inter-observer variations of digital radiograph pulmonary nodule marking by using computer toolkit.

OBJECTIVE: To assess inter-observer variations of pulmonary nodule marking in routine clinical chest digital radiograph (DR) softcopy reading by using a lung nodule computer toolkit. METHODS: A total of 601 chest posterior-anterior DR images were randomly selected from routine outpatient screening in Peking Union Medical College Hospital. Two chest radiologists with experience more than ten years were first asked to read the images and mark all suspicious nodules independently by using computer toolkit IQQA-Chest, and to indicate the likelihood for each nodule detected. They were also asked to draw the boundary of the identified nodule manually on an enlarged region of interest, which was instantly analyzed by IQQA-Chest. Two sets of diagnostic reports, including the marked nodules, likelihood, manually drawn boundaries, quantitative measurements, and radiologists' names, were automatically generated and stored by the computer system. One week later, the two radiologists read the same images together by using the same computer toolkit without referring to their previous reading results. Marking procedure was the same except that consensus was reached for each suspicious region. Statistical analysis tools provided in the IQQA-Chest were used to compare all the three sets of reading results. RESULTS: In the independent readings, Reader 1 detected 409 nodules with a mean diameter of 12.4 mm in 241 patients, and Reader 2 detected 401 nodules with a mean diameter of 12.6 mm in 253 patients. In the consensus reading, a total of 352 nodules with a mean diameter of 12.4 mm were detected in 220 patients. Totally, 42.3% of Reader 1's and 45.1% of Reader 2's marks were confirmed by the consensus reading. About 40% of each reader's marks agreed with the other. There were only 130 (14.4%) out of the total 904 unique nodules were confirmed by both readers and the consensus reading. Moreover, 5.6% (51/904) of the marked regions were rated identical likelihood in all three readings. Statistical analysis showed significant differences between Readers 1 and 2, and between consensus and Reader 2 in determining the likelihood of the marks (P < 0.01), but not between consensus and Reader 1. No significant difference in terms of size was observed in nodule segmentation between either two of the three readings. CONCLUSION: Large variations in nodule marking and nodule-likelihood determination but not in nodule size were observed between experts as well as between single-person reading and consensus reading.

Improved marking and characterizing of pulmonary nodules on digital radiographs using a computer-aided diagnosis system.

OBJECTIVE: To evaluate and reduce inter-observer variations in the detection and characterization of pulmonary nod-ules on digital radiograph (DR) chest images. METHODS: Two hundreds and thirty-two new posterior-anterior DR chest images were collected from out-patient screening patients. Consensus was reached by two experienced radiologists on the marking, rating, and segmentation of small actionable nodules ranged from 5 to 15 mm in diameter using a computer-aided diagnosis (CAD) system. Both their own nodule findings and the computer's automatic nodule detection results were analyzed to make the consensus. Nodules identified together with corresponding likelihood rating and segmentation results were referred as "Gold Standard". Two un-experienced radiologists were asked to first mark and characterize suspicious nodules independently, then were allowed to consult the computer nodule detection results and change their decisions. RESULTS: Large inter-observer variations in pulmonary nodule identification and characterization on DR chest images were observed between un-experienced radiologists. Un-experienced radiologists could greatly benefit from the CAD system, including substantial decrease of inter-observer variation and improvement of nodule detection rates. Moreover, radiologists with different levels of skillfulness could achieve similar high level performance after using the CAD system. CONCLUSION: The CAD system shows a high potential for providing a valuable assistance to the examination of DR chest images.

Discovery of oral fluid biomarkers for human oral cancer by mass spectrometry.

Proteomic analysis of human oral fluid (whole saliva) holds promise as a non-invasive method to identify biomarkers for human oral cancer, a high impact local disease in the oral cavity affecting 38,000 Americans and with 350,000 cases worldwide annually. In this study, matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) was used to profile oral fluid samples from oral cancer and control subjects, and 46 peptides/proteins were found at significantly different levels between the two groups. To identify a candidate protein biomarker, oral fluid samples were separated by liquid chromatography (LC) using a C4 reversed-phase column. The collected LC fractions were monitored by MALDI-MS and the fraction containing the candidate biomarker was digested for LC-MS/MS analysis to identify it. The use of nanospray MS/MS for the identification of candidate peptide biomarkers was also demonstrated. This approach can be useful for the identification of protein or peptide biomarkers following MALDI-MS or surface-enhanced laser desorption/ionization MS profiling of clinical samples. This study clearly demonstrated that oral fluid contains proteomic signatures that may serve as biomarkers for human diseases such as oral cancer. Once discovered and validated on a large and independent clinical cohort, oral fluid proteomic biomarkers may be extensively used for future disease diagnosis.

GPU-friendly marching cubes for visualizing translucent isosurfaces.

Marching cubes has long been employed as a standard indirect volume rendering approach to extract isosurfaces from 3D volumetric data. This paper presents a GPU-friendly MC implementation. Besides the cell indexing, we propose to calculate vertex and normal interpolations by precomputing the expensive equations and looking up these values during runtime. Upon a commodity GPU, our implementation can rapidly extract isosurfaces from a high-resolution volume and render the result. With the proposed parallel marching cubes algorithm, we can naturally generate layer-structured triangles, which facilitate the visualization of multiple-layer translucent isosurfaces without performing computational expensive sorting. The algorithm extracts and draws triangles, in a layer by layer fashion, from back to front.

Virtual acupuncture human based on chinese visible human dataset.

In this paper, we present our application of latest information technology in assisting the Chinese acupuncture research. Having integrated the Chinese Visible Human (CVH) data, virtual reality, visualization and imaging techniques, we have constructed a 3-dimensional digital human model for acupuncture. This model integrates the meridian positioning, acupoint positioning, arbitrary cutting-plane visualization, multi-layer dissection, needle puncturing simulation, as well as the common diseases-therapy information. Our work can be widely applied to Chinese acupuncture education, clinical usage and scientific research.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UNLABELLED: Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex.

Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa alpha7-ring and the intact 690 kDa alpha7beta7beta7alpha7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual alpha-subunits only, and it is generally consistent with the known alpha7beta7beta7alpha7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each beta-subunit. Ion mobility measurements of the alpha7-ring and the alpha7beta7beta7alpha7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.

Differentially expressed protein markers in human submandibular and sublingual secretions.

Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

Creation of the Chinese visible human data set.

The United States Visible Human Project (VHP) created a digital image data set of complete human male (data acquisition finished in November 1994) and female (data acquisition finished in December 1995) cadavers in magnetic resonance imaging (MRI), computed tomography (CT), and anatomical (anatomic serial section) modes. VHP aroused worldwide enthusiasm for Visible Human Research (VHR), and the data set is being used in a variety of research and educational domains. The Visible Korean Human (VKH) male was produced in March 2001. To accelerate worldwide VHR and to promote virtual anatomy as a revolutionary break with conventional anatomy, more visible human data sets representative of different populations of the world are in demand. The Chinese Visible Human (CVH) male (created in October 2002) and female (created in February 2003) project achieved greater integrity of images, easier blood vessel identification, and were free of organic lesion (unlike the other visible human projects). We performed data acquisition, three-dimensional (3D) reconstruction, and visualization with improved technology to create CVH male and female. CVH is the first volumetric data representing a complete normal adult human male and female of an Asian population. This article presents the history of Chinese Visible Human cadavers and the methods and technology used to produce the data set.