methods-for-characterizing-co-development-biofilm-habitat

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Northwestern University

There is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization of 3-nitrotyrosine (after reduction to 3-amino-tyrosine) and 3,4-dihydroxyphenylalanine with benzylamine derivatives to yield 6-amino- and 6-benzylamine-substituted benzoxazoles, which display characteristic fluorescence properties. The methodology can be expanded to other substituted benzylamines, which carry functional groups for affinity enrichment.

Selective fluorogenic derivatization of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine in peptides: a method designed for quantitative proteomic analysis.

Hydraulic conductivity identification remains a challenging inverse problem in ground water modeling because of the inherent nonuniqueness and lack of flexibility in parameterization methods. This study introduces maximum weighted log-likelihood estimation (MWLLE) along with multiple generalized parameterization (GP) methods to identify hydraulic conductivity and to address nonuniqueness and inflexibility problems in parameterization. A scaling factor for information criteria is suggested to obtain reasonable weights of parameterization methods for the MWLLE and model averaging method. The scaling factor is a statistical parameter relating to a desired significance level in Occam's window and the variance of the chi-squares distribution of the fitting error. Through model averaging with multiple GP methods, the conditional estimate of hydraulic conductivity and its total conditional covariances are calculated. A numerical example illustrates the issue arising from Occam's window in estimating model weights and shows the usefulness of the scaling factor to obtain reasonable model weights. Moreover, the numerical example demonstrates the advantage of using multiple GP methods over the zonation and interpolation methods because GP provides better models in the model averaging method. The methodology is applied to the Alamitos Gap area, California, to identify the hydraulic conductivity field. The results show that the use of the scaling factor is necessary in order to incorporate good parameterization methods and to avoid a dominant parameterization method.

Multiple parameterization for hydraulic conductivity identification.

The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.

Hydrogen isotopic profile in the characterization of sugars. Influence of the metabolic pathway.

Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.

Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

This study examined students' conceptions about the equal sign in light of historical findings with an international comparison group. Textbooks for preparation of students as mathematics teachers were examined. Participants were sixth-grade students from the USA (n = 105) and China (n = 145). About 98% of the Chinese children correctly answered all items by providing conceptually accurate explanations, but only 28% of the U.S. sample did. Textbooks for education majors who would teach in the USA rarely discussed the equal sign as equivalence while the Chinese texts introduced the "equal sign" in a context of relationships and discussed it as "balance," "sameness," or "equivalence."

Thirty years of research: interpretations of the equal sign in China and the USA.

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.

Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

Carbazole, one representative of non-alkaline nitrogen heterocyclic compounds, is widespread in the natural environment and harmful to human health. In this research, the seven bacterial strains using carbazole as their sole carbon, nitrogen and energy source were isolated from activated sludge of a coking wastewater treatment plant. All strains efficiently degraded 500 mg/L of carbazole in the medium within 36 h. Based on the DNA sequence and phylogenetic tree analysis, the seven strains were identified as the genera Pseudomonas with different evolutionary pathways. PCR analysis revealed that the seven isolates carried the car gene. Moreover, all of these strains could utilize and transform ammonium and nitrate efficiently, and the six strains except BC043 strain coded the nitrite reductase gene (nirS) and the nitrous oxide reductase (nosZ), that indicated their denitrification ability. All these strains may be useful in the bioremediation of environments contaminated by carbazole.

Biodegradation of carbazole by the seven Pseudomonas sp. strains and their denitrification potential.

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

Gastric cancer is a common malignancy with high mortality rate, and surgical resection is the primary treatment.

Multivariate analysis of prognostic factors in 549 patients undergoing surgical treatment of gastric cancer.

Quantum capacitance is a fundamental quantity that can directly reveal many-body interactions among electrons and is expected to play a critical role in nanoelectronics. One of the many tantalizing recent physical revelations about quantum capacitance is that it can possess a negative value, hence allowing for the possibility of enhancing the overall capacitance in some particular material systems beyond the scaling predicted by classical electrostatics. Using detailed quantum mechanical simulations, we found an intriguing result that mechanical strains can tune both signs and values of quantum capacitance. We used a small coaxially gated carbon nanotube as a paradigmatical capacitor system and showed that, for the range of mechanical strain considered, quantum capacitance can be adjusted from very large positive to very large negative values (in the order of plus/minus hundreds of attofarads), compared to the corresponding classical geometric value (0.31035 aF). This finding opens novel avenues in designing quantum capacitance for applications in nanosensors, energy storage, and nanoelectronics.

Xiaobao Li

Department of Civil and Environmental Engineering

Northwestern University

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity