A hybrid electronic nose system for discrimination of pathogenic bacterial volatile compounds.

A hybrid electronic nose comprising an array of three organic-inorganic nanocomposite gas sensors [zinc tetra tert-butyl phthalocyanine (ZnTTBPc), zinc tetra-phenyl porphyrin (ZnTPP), and cobalt tetraphenyl-porphyrin (CoTPP)] coupled with three commercial metal-oxide semiconductor gas sensors (TGS 2444, TGS 2603 and TGS 2620) was developed to discriminate bacterial volatile compounds. Each type of gas sensor had its own strengths and weaknesses in terms of its capability to detect complex odors from the five different bacterial species tested. Bacterial samples were controlled at a fixed initial bacterial concentration by measuring the optical density at 600 nm of the culture suspensions. A comparative evaluation of the volatile compound fingerprints from five bacterial species grown in Luria-Bertani medium was conducted to identify the optimal incubation time for detection of volatile biomarkers to discriminate among bacteria. The results suggest that the hybrid electronic nose was indeed able to discriminate among the bacterial species and culture media, with a variance based on contributions of 92.4% from PC1 and 7.2% from PC2, at an incubation time of 6 hours. Furthermore, the results of hierarchical cluster analysis showed that bacterial odor data formed two major bacterial groups, with the maximum cluster distance close to 25. Intra-group similarity was demonstrated as the two bacterial species (E. cloacae and P. aeruginosa) from among the Gram-negative bacteria had a greater similarity with a cluster distance close to 4. Finally, the minimum distance between E. cloacae and S. Typhi was approximately 1, at an equal distance from E. coli and S. aureus.

Roles of kininogen-1, basement membrane specific heparan sulfate proteoglycan core protein, and roundabout homolog 4 as potential urinary protein biomarkers in diabetic nephropathy.

Diabetic nephropathy, a major complication of diabetes mellitus (DM), is increasing worldwide and the large majority of patients have type 2 DM. Microalbuminuria has been used as a diagnostic marker of diabetic nephropathy. But owing to its insufficient sensitivity and specificity, other biomarkers are being sought. In addition, the pathophysiology of diabetic nephropathy is not fully understood and declines in renal function occur even without microalbuminuria. In this study, we investigated urinary proteins from three study groups (controls, and type 2 diabetic subjects with or without microalbuminuria). Non-targeted label-free Nano-LC QTOF analysis was conducted to discover underlying mechanisms and protein networks, and targeted label-free Nano-LC QTOF with SWATH was performed to qualify discovered protein candidates. Twenty-eight proteins were identified as candidates and functionally analyzed via String DB, gene ontology and pathway analysis. Four predictive mechanisms were analyzed: i) response to stimulus, ii) platelet activation, signaling and aggregation, iii) ECM-receptor interaction, and iv) angiogenesis. These mechanisms can provoke kidney dysfunction in type 2 diabetic patients via endothelial cell damage and glomerulus structural alteration. Based on these analyses, three proteins (kininogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, and roundabout homolog 4) were proposed for further study as potential biomarkers. Our findings provide insights that may improve methods for both prevention and diagnosis of diabetic nephropathy.

Development of Organic-Inorganic Hybrid Optical Gas Sensors for the Non-Invasive Monitoring of Pathogenic Bacteria.

Hybrid optical gas sensors, based on different organic and inorganic materials, are proposed in this paper, with the aim of using them as optical artificial nose systems. Three types of organic and inorganic dyes, namely zinc-porphyrin, manganese-porphyrin, and zinc-phthalocyanine, were used as gas sensing materials to fabricate a thin-film coating on glass substrates. The performance of the gas sensor was enhanced by a thermal treatment process. The optical absorption spectra and morphological structure of the sensing films were confirmed by UV-Vis spectrophotometer and atomic force microscope, respectively. The optical gas sensors were tested with various volatile compounds, such as acetic acid, acetone, ammonia, ethanol, ethyl acetate, and formaldehyde, which are commonly found to be released during the growth of bacteria. These sensors were used to detect and discriminate between the bacterial odors of three pathogenic species (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) grown in Luria-Bertani medium. Based on a pattern recognition (PARC) technique, we showed that the proposed hybrid optical gas sensors can discriminate among the three pathogenic bacterial odors and that the volatile organic compound (VOC) odor pattern of each bacterium was dependent on the phase of bacterial growth.

Metal complexation by histidine-rich peptides confers protective roles against cadmium stress in Escherichia coli as revealed by proteomics analysis.

The underlying mechanism and cellular responses of bacteria against toxic cadmium ions is still not fully understood. Herein, Escherichia coli TG1 expressing hexahistidine-green fluorescent protein (His6GFP) and cells expressing polyhistidine-fused to the outer membrane protein A (His-OmpA) were applied as models to investigate roles of cytoplasmic metal complexation and metal chelation at the surface membrane, respectively, upon exposure to cadmium stress. Two-dimensional gel electrophoresis (2-DE) and two-dimensional difference in gel electrophoresis (2D-DIGE) in conjunction with mass spectrometry-based protein identification had successfully revealed the low level expression of antioxidative enzymes and stress-responsive proteins such as manganese-superoxide dismutase (MnSOD; +1.65 fold), alkyl hydroperoxide reductase subunit C (AhpC; +1.03 fold) and DNA starvation/stationary phase protection protein (Dps; -1.02 fold) in cells expressing His6GFP in the presence of 0.2 mM cadmium ions. By contrarily, cadmium exposure led to the up-regulation of MnSOD of up to +7.20 and +3.08 fold in TG1-carrying pUC19 control plasmid and TG1 expressing native GFP, respectively, for defensive purposes against Cd-induced oxidative cell damage. Our findings strongly support the idea that complex formation between cadmium ions and His6GFP could prevent reactive oxygen species (ROS) caused by interaction between Cd2+ and electron transport chain. This coincided with the evidence that cells expressing His6GFP could maintain their growth pattern in a similar fashion as that of the control cells even in the presence of harmful cadmium. Interestingly, overexpression of either OmpA or His-OmpA in E. coli cells has also been proven to confer protection against cadmium toxicity as comparable to that observed in cells expressing His6GFP. Blockage of metal uptake as a consequence of anchored polyhistidine residues on surface membrane limited certain amount of cadmium ions in which some portion could pass through and exert their toxic effects to cells as observed by the increased expression of MnSOD of up to +9.91 and +3.31 fold in case of TG1 expressing only OmpA and His-OmpA, respectively. Plausible mechanisms of cellular responses and protein mapping in the presence of cadmium ions were discussed. Taken together, we propose that the intracellular complexation of cadmium ions by metal-binding regions provides more efficiency to cope with cadmium stress than the blockage of metal uptake at the surface membrane. Such findings provide insights into the molecular mechanism and cellular adaptation against cadmium toxicity in bacteria.

Oxidative responses and defense mechanism of hyperpigmented P. aeruginosa as characterized by proteomics and metabolomics.

Pseudomonas aeruginosa is known to produce multiple types of pigment which are involved in its pathogenicity and survival in certain environments. Herein, we reported the identification of P. aeruginosa dark-brown hyperpigmented (HP) strains which have been isolated from clinical samples. In order to study the role of these dark-brown containing secretions, alterations of metabolic processes and cellular responses under microenvironment of this bacterial pathogen, two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF) were performed. Protein spots showing the most significant differences and high spot optical density values were selected for further characterization. Fold difference of protein expression levels among those spots were calculated. Three major groups of proteins including anti-oxidant enzyme such as catalase, alkyl hydroperoxide reductase and also iron-superoxide dismutase (Fe-SOD), transmembrane proteins as well as proteins involved in energy metabolism such as ATP synthase and pyruvate/2-oxoglutarate dehydrogenase were significantly decreased in P. aeruginosa HP. Whereas, malate syntase and isocitrate lyase, the key enzyme in glyoxylate cycle as well as alcohol dehydrogenase were significantly increased in P. aeruginosa HP, as compared to the reference strain ATCC 27853. Moreover, the HP exerted SOD-like activity with its IC50 equal to 0.26 mg/ml as measured by NBT assay. Corresponding to secretomic metabolome identification, elevated amounts of anti-oxidant compounds are detected in P. aeruginosa HP than those observed in ATCC 27853. Our findings indicated successful use of proteomics and metabolomics for understanding cell responses and defense mechanisms of P. aeruginosa dark-brown hyperpigmented strains upon surviving in its microenvironment.

Proteomic and bioinformatic discovery of biomarkers for diabetic nephropathy.

Diabetes is associated with numerous metabolic and vascular risk factors that contribute to a high rate of micro-vascular and macro-vascular disorders leading to mortality and morbidity from diabetic complications. In this case, the major cause of death in overall diabetic patients results from diabetic nephropathy (DN) or renal failure. The risk factors and mechanisms that correspond to the development of DN are not fully understood and so far, no specific and sufficient diagnostic biomarkers are currently available other than micro- or macroalbuminuria. Therefore, this review describes current and novel protein biomarkers in the context of DN as well as probable proteins biomarkers associated with pathological processes for the early stage of DN via proteomics data together with bioinformatics. In addition, the mechanisms involved in early development of diabetic vascular disorders and complications resulting from glucose induced oxidative stress will also be explored.

Novel role of 4-hydroxy-2-nonenal in AIFm2-mediated mitochondrial stress signaling.

Cardiovascular complications are major side effects of many anticancer drugs. Accumulated evidence indicates that oxidative stress in mitochondria plays an important role in cardiac injury, but how mitochondrial redox mechanisms are involved in cardiac dysfunction remains unclear. Here, we demonstrate that 4-hydroxy-2-nonenal (HNE) activates the translocation of the mitochondrial apoptosis inducing factor (AIFm2) and facilitates apoptosis in heart tissue of mice and humans. Doxorubicin treatments significantly enhance cardiac levels of HNE and AIFm2. HNE adduction of AIFm2 inactivates the NADH oxidoreductase activity of AIFm2 and facilitates its translocation from mitochondria. His 174 on AIFm2 is the critical target of HNE adduction that triggers this functional switch. HNE adduction and translocation of AIFm2 from mitochondria upon Doxorubicin treatment are attenuated by superoxide dismutase mimetics. These results identify a previously unrecognized role of HNE with important consequences for mitochondrial stress signaling, heart failure, and the side effects of cancer therapy.

Retinol-binding protein 4 and its potential roles in hypercholesterolemia revealed by proteomics.

Effects of hypercholesterolemia on alterations of serum proteins have not been fully elucidated. Herein, using two-dimensional gel electrophoresis (2-DE) in conjunction with LC-MS searching has successfully been carried out to investigate the change of protein expression profiles as consequences of raised blood cholesterol at different levels (normal group: total cholesterol 200 mg/dL; borderline high group: total cholesterol 200-239 mg/dL; and high group: total cholesterol ≥ 240 mg/dL) (n = 45). Results revealed that down-regulation of retinol-binding protein 4 (RBP4) (-2.26 fold), transthyretin (-1.25 fold) and gelsolin (-1.47 fold) was observed in the high group. Meanwhile, the other proteins such as haptoglobin, complement factor B and CD5 antigen-like protein were up-regulated upto +3.24, +1.96 and +2.04 fold, respectively. Confirmation by Western blotting revealed a significant reduction of RBP4 (approximately 50 %) in individual samples derived from the high group. Presumptive conclusion can be drawn that down-regulation of RBP4 might be attributable to the inflammation of adipocytes caused by the release of proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1ß) from adipose tissues. Moreover, the decrease of transthyretin might also be taken into accounts since it is known that the transthyretin usually forms complex with RBP4 to prevent glomerular filtration and excretion through the kidney. The suppressing effect on RBP4 should be potentiated by the increase of complement factor B and CD5 antigen-like protein, which rendered the adipose tissues to overwhelm the liberation of RBP4 to blood circulation by metabolic and inflammatory processes. Such inflammation could further modulate the induction of cytokine release (e.g. IL-6 and IL-1ß), resulting in the synthesis of acute phase protein, in particular, haptoglobin and C-reactive proteins from hepatocytes. However, the mechanism of gelsolin reduction remains unclear. Among these differentially expressed proteins, the RBP4 has been proposed as a major linkage between hypercholesterolemia, adipose tissues, liver and kidney, which is believed to be a potential biomarker for metabolic and cardiovascular disorders associated with dyslipidemia in the future.

Proteomic alterations of Escherichia coli by paraquat.

Paraquat (PQ; a widely used herbicide) exerts its harmful effect to human, mammals and microorganisms upon intracellular conversion to superoxide radical. Cellular responses against toxic paraquat remain not fully understood, especially on the adaptive metabolic changes as a consequence of oxidative burden. In this study, alterations of metabolic processes of Escherichia coli (E. coli) by paraquat were systematically investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF). In host cells, the first line mechanism was scrutinized by a remarkable induction of endogenous superoxide dismutase (E. coli SOD). The second line involved in the metabolic adaptation and compensation for energy production by up- or down-regulation of the enzymes implicated in glycolysis and tricarboxylic acid cycle. Notably, down-regulation of aconitase enzyme and changes of enzyme isoform from the acidic (pI~5.29) to the higher basidic form (pI~5.59) were detected. Meanwhile, up-regulation of fumarase approximately 4-5 folds were observed. Importantly, overexpression of human manganese superoxide dismutase (human Mn-SOD) in E. coli cells could in turn down-regulate the expression of fumarase enzyme. This observation was not found when the cells expressing human catalase were tested. Other mechanisms such as changes of purine nucleoside phosphorylase and protein transporters (D-ribose-binding protein and oligopeptide binding protein) were also accounted. However, among all the differentially expressed proteins, the fumarase enzyme is evidenced to be a major target responsible for superoxide-generating paraquat, which may further be applied as a potential biomarker for paraquat toxicity in the future.

Modeling the LPS neutralization activity of anti-endotoxins.

Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural components of the outer membrane of Gram-negative bacteria that serve as a barrier and protective shield between them and their surrounding environment. LPS is considered to be a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines which mediate the host immune response and culminating in septic shock. Quantitative structure-activity relationship studies of the LPS neutralization activities of anti-endotoxins were performed using charge and quantum chemical descriptors. Artificial neural network implementing the back-propagation algorithm was selected for the multivariate analysis. The predicted activities from leave-one-out cross-validation were well correlated with the experimental values as observed from the correlation coefficient and root mean square error of 0.930 and 0.162, respectively. Similarly, the external testing set also yielded good predictivity with correlation coefficient and root mean square error of 0.983 and 0.130. The model holds great potential for the rational design of novel and robust compounds with enhanced neutralization activity.