SUMMARY Bacterial biofilms in the colon alter the host tissue microenvironment. A role for biofilms in colon cancer metabolism has been suggested but to date has not been evaluated. Using metabolomics, we investigated the metabolic influence that microbial biofilms have on colon tissues and the related occurrence of cancer. Patient-matched colon cancers and histologically normal tissues, with or without biofilms, were examined. We show the upregulation of polyamine metabolites in tissues from cancer hosts with significant enhancement of N1, N12-diacetylspermine in both biofilm positive cancer and normal tissues. Antibiotic treatment, which cleared biofilms, decreased N1, N12-diacetylspermine levels to those seen in biofilm negative tissues, indicating that host cancer and bacterial biofilm structures contribute to the polyamine metabolite pool. These results show that colonic mucosal biofilms alter the cancer metabolome, to produce a regulator of cellular proliferation and colon cancer growth potentially affecting cancer development and progression.

Contrary to the widely accepted mechanism of the Hajos-Parrish-Eder-Sauer-Wiechert reaction, we have obtained evidence for the involvement of only one proline molecule in the transition states of both inter- and intramolecular aldol reactions. Our conclusions are based on kinetic measurements and the absence of nonlinear and dilution effects on the asymmetric catalysis, and are supported by B3LYP/6-31G* calculations. Complementary to recent theoretical studies, our results provide the foundation of a unified enamine catalysis mechanism of proline-catalyzed inter- and intramolecular aldol reactions.

The mechanism of the proline-catalyzed aldol reaction has stimulated considerable debate, and despite limited experimental data, at least five different mechanisms have been proposed. Complementary to recent theoretical studies we have initiated an experimental program with the goal of clarifying some of the basic mechanistic questions concerning the proline-catalyzed aldol reaction. Here we summarize our discoveries in this area and provide further evidence for the involvement of enamine intermediates. D iscovered in the early 1970s, the Hajos-Parrish-EderSauer-Wiechert reaction (1, 2), a proline-catalyzed intramolecular aldol reaction, represents not only the first asymmetric aldol reaction invented by chemists but also the first highly enantioselective organocatalytic transformation [1(4) 3 2(5) 3 3(6)] (Eq. 1 of Scheme 1) (3-6). Inspired by Nature's phenomenal enzymes, which catalyze direct asymmetric aldolizations of unmodified carbonyl compounds (7, 8), we have recently extended the Hajos-Parrish-Eder-Sauer-Wiechert reaction to the first intermolecular variant (7 ϩ 8 3 9) (Eq. 2 of Scheme 1) (9), and to several other reactions including prolinecatalyzed asymmetric Mannich (10), Michael (11), ␣-amination (12), and intramolecular enolexo aldolization reactions (10 3 11) (13) (Eq. 3 of Scheme 1) (14-18).Similar to the aldolase enzymes, proline catalyzes direct asymmetric aldol reactions between two different carbonyl compounds to provide aldol products in excellent yields and enantioselectivities. Early on it has been speculated that in addition to operating on related substrates, both class I aldolases and proline may also share a similar enamine mechanism (19,20). However, there has been some debate over several mechanistic aspects of the reaction, and a number of alternative models have been proposed. For example, Hajos (1) suggested a mechanism that involves the ''activation'' of one of the enantiotopic acceptor carbonyl groups as a carbinol amine (A of Scheme 1). At least the stereochemistry of this model was questioned by Jung (19) soon after its initial proposal. An enamine mechanism was suggested by various groups already in the 1970s and 1980s (19-21). Nonlinearity studies by Agami and colleagues (21) have led to the proposal of a side-chain enamine mechanism that involves two proline molecules in the C-C-bondforming transition state, one engaged in enamine formation and the other as a proton transfer mediator (B of Scheme 1). Swaminathan et al. (22) favor a heterogeneous aldolization mechanism on the surface of crystalline proline (C of Scheme 1), despite the fact that many proline-catalyzed aldolizations are completely homogenous. Agami's widely accepted twoproline mechanism was recently challenged when we proposed a homogenous one-proline enamine mechanism for the intermolecular variant in which the various proton transfers are mediated by proline's carboxylic acid functionality (9). On the basis of density functional theory calculations, subsequently proposed a very similar mechanism for the in...

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year.

We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorousphase interactions. This ''soft'' immobilization allows efficient desorption/ionization while also enabling the use of surfacewashing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing ␤-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65°C and 5.5, respectively, and the activity was inhibited by both phenylethyl-␤-D-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced ␥-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis.fluorous phase ͉ microbial community ͉ carbohydrate

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II proteinarginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online (). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

BackgroundRapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.ResultsWe have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control.ConclusionsSimple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems.