Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor.

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

Neighboring π-Amide Participation in Thioether Oxidation: Conformational Control.

The electrochemical oxidation of thioethers is shown to be facilitated by neighboring amide participation. (1)H NMR spectroscopic analysis in acetonitrile solution of two conformationally constrained compounds with such facilitation shows that two-electron participation by the amide π2 orbital can occur to stabilize the developing sulfur radical cation.

Potential applications of induced pluripotent stem cells (iPSCs) in hepatology research.

Induced pluripotent stem cells (iPSCs) share many characteristics with embryonic stem cells (ESCs), but circumvent most of the ethical issues surrounding ESCs. The use of iPSCs to treat liver diseases is gaining increasing interest. Recent studies show these iPSCs can be differentiated into the hepatic lineage and provide an accurate model for liver diseases, drug screening and drug toxicity testing. Recently, the potential application of iPSC-derived hepatocytes to be used in cell-based therapies has been explored as a novel strategy to treat human liver disease. However, the successful use of these iPSC-derived hepatocytes hinges on overcoming the inherent problems of using iPSC in cell-based therapies. Given these problems are addressed in the future, these iPSC derived hepatocytes provide a limitless supply of cells that could be used to treat liver diseases not only in screening and toxicity testing but also in cell-based therapies.

An electrochemical aptasensor for thrombin using synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructures for signal amplification.

In this work, a sensitive electrochemical aptasensor for thrombin (TB) based on synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructure has been constructed. With the advantages of large surface area and outstanding catalytic performance, porous Au@Pd core-shell nanostructures were firstly employed as the nanocarrier for the immobilization of electroactive toluidine blue (Tb), hemin/G-quadruplex formed by intercalating hemin into the TB aptamer (TBA) and glucose oxidase (GOx). As a certain amount of glucose was added into the detection cell, GOx rapidly catalyzed the oxidation of glucose, coupling with the local generation of H2O2 in the presence of dissolved O2. Then, porous Au@Pd nanoparticles and hemin/G-quadruplex as the peroxidase mimics efficiently catalyzed the reduction of H2O2, amplifying the electrochemical signal and improving the sensitivity. Finally, a detection limit of 0.037pM for TB was achieved. The excellent performance of the aptasensor indicated its promising prospect as a valuable tool in simple and cost-effective TB detection in clinical application.

Synergistic effects of IAP inhibitor LCL161 and paclitaxel on hepatocellular carcinoma cells.

Inhibitor of Apoptosis Proteins (IAPs) are key regulators of apoptosis in hepatocellular carcinoma (HCC) and their expression is negatively correlated with patient survival. LCL161 is a small molecule inhibitor of IAPs that has potent antitumour activity in a range of solid tumours. In HCC, response to LCL161 therapy has shown to be mediated by Bcl-2 expression. In this study, we aim to determine whether LCL161 has any therapeutic potential in HCC. Protein expression was determined by Western blot. Cell proliferation was determined by Cell Proliferation ELISA and BrdU colorimetric assays. Apoptosis was determined by Annexin V assay. Cell cycle analysis was performed by staining cells with propidium iodide and analysed in a FACScan. Automated Cell Counter and phase contrast microscopy were used to determine the cell viability. We have found that LCL161 targets (cIAP1, cIAP2 and XIAP) were up-regulated in HCC tumours. Both high Bcl-2 expressing HuH7 cells and low Bcl-2 expressing SNU423 cells showed strong resistance to LCL161 therapy with significant effects on both apoptosis and cell viability only evident at LCL161 concentrations of ⩾100µM. At these doses there was significant inhibition of IAP targets, however there was also significant inhibition of off-target proteins including pERK and pJNK suggesting apoptosis caused by drug toxicity. However, when used in combination with paclitaxel in HuH7 and SNU423 cells, LCL161 had significant antiproliferative effects at doses as low as 2µM and this was independent of Bcl-2 expression. Thus, LCL161 may be a useful agent in combination with paclitaxel to treat liver tumours.

Physical and chemical insults induce inflammation and gastrointestinal cancers.

Chronic inflammation associated with viral and bacterial infections of the gastrointestinal tract (GI) and liver renders these organs susceptible to tumour development. There is also a growing body of evidence demonstrating that chemical and physical insults promote GI cancers by inducing inflammation. For example, excessive alcohol consumption and tobacco smoking induces inflammation and gastrointestinal carcinogenesis. Likewise, drinking hot beverages and intentional or accidental exposure to toxic substances leads to inflammation and GI cancer formation. However, further work needs to be undertaken using animal models to separate the direct carcinogenic effects of physical and chemical insults from the indirect effects of these insults to promote tumor formation through tissue inflammation.

Tumoricidal effects of the JAK inhibitor Ruxolitinib (INC424) on hepatocellular carcinoma in vitro.

Hepatocellular carcinoma (HCC) is an aggressive tumour with limited treatment options. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signalling pathway plays a key role in promoting tumorigenesis in HCC. Recently a new JAK inhibitor Ruxolitinib (INC424) has been developed by Novartis Pharmaceuticals and it shows high affinity for JAK signalling with very low affinity for non-JAK targets. Clinical trials have demonstrated that Ruxolitinib has good therapeutic efficacy for the treatment of myelofibrosis and is currently FDA approved for the treatment of advanced stages of this disease. Our study tested the effects of Ruxolitinib on HCC tumorigenesis in vitro. Ruxolitinib effectively inhibited JAK/STAT signalling in HCC cells with a significant reduction in the expression of JAK downstream targets pSTAT1 and pSTAT3. Ruxolitinib also caused a marked reduction in the proliferation and colony formation of HCC cells. The antiproliferative effect of Ruxolitinib on HCC cells is unlikely due to off-target effects with no inhibition of key regulators of other cell proliferative pathways. To our knowledge this study is the first to report on the effect of Ruxolitinib on liver cancer cells.

Efficacy of using cancer stem cell markers in isolating and characterizing liver cancer stem cells.

Recent evidence suggests that a subset of hepatocellular carcinomas (HCCs) are derived from liver cancer stem cells (LCSCs). In order to isolate and characterize LCSCs, reliable markers that are specific to these cells are required. We evaluated the efficacy of a range of cancer stem cell (CSC) markers in isolating and characterizing LCSCs. We show that the most widely used CSC markers are not specific to LCSCs. By western analysis, protein expression of the common markers showed no significant difference between HCC tumor tissues and adjacent non-cancerous liver. Further, isolation of LCSCs from common HCC cell lines using FACScan and microbeads showed no consistent marker expression pattern. We also show that LCSCs have unique subtypes. Immunohistochemistry of HCC tissues showed that different HCCs express unique combinations of LCSC markers. Quantitative real-time polymerase chain reaction analysis showed that LCSCs isolated using different markers in the same HCC phenotype had different expression profiles. Likewise, LCSCs isolated from different HCC phenotypes with the same marker also had unique expression profiles and displayed varying resistance profiles to Sorafenib. Thus, using a range of commonly used CSC markers in HCCs and cell lines, we demonstrate that currently available markers are not specific for LCSCs. LCSCs have unique subtypes that express distinctive combinations of LCSC markers and altered drug resistance profiles, making their identification problematic.

Lactate as a biomarker for sleep.

STUDY OBJECTIVES: An ideal biomarker for sleep should change rapidly with sleep onset, remain at a detectably differential level throughout the sleep period, and exhibit a rapid change with waking. Currently, no molecular marker has been identified that exhibits all three properties. This study examined three substances (lactate, glucose, and glutamate) for suitability as a sleep biomarker. DESIGN: Using amperometric biosensor technology in conjunction with electroencephalograph (EEG) and electromyograph (EMG) monitoring, extracellular concentrations of lactate and glucose (Cohort 1) as well as lactate and glutamate (Cohort 2) were recorded over multiple sleep/wake cycles. PATIENTS OR PARTICIPANTS: There were 12 C57Bl/6J male mice (3-5 mo old). INTERVENTIONS: Sleep and waking transitions were identified using EEG recordings. Extracellular concentrations of lactate, glucose, and glutamate were evaluated before and during transition events as well as during extended sleep and during a 6-h sleep deprivation period. MEASUREMENTS AND RESULTS: Rapid and sustained increases in cortical lactate concentration (approximately 15 µM/min) were immediately observed upon waking and during rapid eye movement sleep. Elevated lactate concentration was also maintained throughout a 6-h period of continuous waking. A persistent and sustained decline in lactate concentration was measured during nonrapid eye movement sleep. Glutamate exhibited similar patterns, but with a much slower rise and decline (approximately 0.03 µM/min). Glucose concentration changes did not demonstrate a clear correlation with either sleep or wake. CONCLUSIONS: These findings indicate that extracellular lactate concentration is a reliable sleep/wake biomarker and can be used independently of the EEG signal.

Neighboring pyrrolidine amide participation in thioether oxidation. Methionine as a "hopping" site.

Methionine residues have been shown to function as efficient "hopping" sites in long-range electron transfer in model polyprolyl peptides. We suggest that a key to this ability of methionine is stabilization of the transient sulfur radical cation by neighboring proline amide participation. That is, in a model system a neighboring pyrrolidine amide lowers the oxidation potential of the thioether by over 0.5 V by formation of a two-center three-electron SO bond.

Antimicrobial activity of surface attached marine bacteria in biofilms.

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an 'inducer' bacterium stimulated the production of antimicrobial molecules by 'producer' bacteria that were active against the 'inducer' bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.

Simultaneous real-time measurement of EEG/EMG and L-glutamate in mice: A biosensor study of neuronal activity during sleep.

We report on electroencephalograph (EEG) and electromyograph (EMG) measurements concurrently with real-time changes in L-glutamate concentration. These data reveal a link between sleep state and extracellular neurotransmitter changes in a freely-moving (tethered) mouse. This study reveals, for the first time in mice, that the extracellular L-glutamate concentration in the pre-frontal cortex (PFC) increases during periods of extended wakefulness, decreases during extended sleep episodes and spikes during periods of REM sleep. Individual sleep epochs (10 s in duration) were scored as wake, slow-wave (SW) sleep or rapid eye movement (REM) sleep, and then correlated as a function of time with measured changes in L-glutamate concentrations. The observed L-glutamate levels show a statistically significant increase of 0.86 ± 0.26 µM (p < 0.05) over 37 wake episodes recorded from all mice (n = 6). Over the course of 49 measured sleep periods longer than 15 min, L-glutamate concentrations decline by a similar amount (0.88 ± 0.37 µM, p < 0.08). The analysis of 163 individual REM sleep episodes greater than one min in length across all mice (n = 6) demonstrates a significant rise in L-glutamate levels as compared to the 1 min preceding REM sleep onset (RM-ANOVA, DF = 20, F = 6.458, p < 0.001). The observed rapid changes in L-glutamate concentration during REM sleep last only between 1 and 3 min. The approach described can also be extended to other regions of the brain which are hypothesized to play a role in sleep. This study highlights the importance of obtaining simultaneous measurements of neurotransmitter levels in conjunction with sleep markers to help elucidate the underlying physiological and ultimately the genetic components of sleep.

Heterogeneity of surface attached microbial communities from Sydney Harbour, Australia.

There is limited information on bacterial communities attached to marine surfaces. These surface attached bacterial communities can vary at a micro scale and these differences may be due to surface characteristics in marine environments. The current study investigates the heterogeneity of bacterial communities on five different marine invertebrates (Heliocidaris erythrogramma, Austrocochlea concamerata, Crassostrea gigas, Dendrilla rosea, and Actinia tenebrosa), the alga, Lobophora variegata and marine gravel from a 20 m × 20 m quadrant in Camp Cove, Sydney Harbour, Australia. Terminal restriction fragment length polymorphism (TRFLP) of 16S sequences showed that each surface contained unique combinations of TRFLP fragment lengths. Phylogenetic analysis of random clones picked from clone libraries constructed from the amplified 16S sequences revealed that 16S sequences from the communities on different surfaces clustered into distinct clades. None of the bacteria identified by 16S sequencing of the whole (uncultured) microbial communities was detected after cultivation. Overall, the study shows surface type plays a major role in shaping microbial communities in marine environments.

Diversity and antimicrobial activities of surface-attached marine bacteria from Sydney Harbour, Australia.

Marine bacteria are a rich source of potentially useful antimicrobial molecules. However, much of the microbial diversity in marine ecosystems with its potential for uncovering new antimicrobial compounds remains to be discovered. This is particularly true for surface-attached marine bacteria, which comprise microbial communities that are generally unique to a host surface and geographic location. The current study characterises culturable microbial communities on marine surfaces from Sydney Harbour, Australia, and tests their antimicrobial activities. A high proportion (47%) of the 104 marine isolates from Sydney Harbour could not be classified to a known genus based on 16S ribosomal RNA gene sequences. Assays of antimicrobial activity from the 104 isolates showed that antimicrobial production is not widespread throughout the phylogeny of isolates with 8 of the 10 antimicrobial producers clustering into a distinct phylogenetic clade. These 8 closely related antibacterial isolates had potent activity in antibacterial cross-dilution assays, with no growth of target bacteria at supernatant concentrations of less than 6.6% v/v. To gain an insight into the types of molecules responsible for this potent activity, differential polarity extractions were carried out on antibacterial culture supernatants from these 8 isolates. All of the activity fractionated into the most polar phase, suggesting that the antibacterial molecules are highly polar. Proteolytic digestion inhibited activity, indicating that the antibacterial molecules were proteins. This study is the first to link the phylogeny of numerous surface-attached marine bacteria with antimicrobial production.

Artificial neural network--based analysis of high-throughput screening data for improved prediction of active compounds.

Artificial neural networks (ANNs) are trained using high-throughput screening (HTS) data to recover active compounds from a large data set. Improved classification performance was obtained on combining predictions made by multiple ANNs. The HTS data, acquired from a methionine aminopeptidases inhibition study, consisted of a library of 43,347 compounds, and the ratio of active to nonactive compounds, R(A/N), was 0.0321. Back-propagation ANNs were trained and validated using principal components derived from the physicochemical features of the compounds. On selecting the training parameters carefully, an ANN recovers one-third of all active compounds from the validation set with a 3-fold gain in R(A/N) value. Further gains in R(A/N) values were obtained upon combining the predictions made by a number of ANNs. The generalization property of the back-propagation ANNs was used to train those ANNs with the same training samples, after being initialized with different sets of random weights. As a result, only 10% of all available compounds were needed for training and validation, and the rest of the data set was screened with more than a 10-fold gain of the original R(A/N) value. Thus, ANNs trained with limited HTS data might become useful in recovering active compounds from large data sets.

Neighboring amide participation in thioether oxidation: relevance to biological oxidation.

To investigate neighboring amide participation in thioether oxidation, which may be relevant to brain oxidative stress accompanying beta-amyloid peptide aggregation, conformationally constrained methylthionorbornyl derivatives with amido moieties were synthesized and characterized, including an X-ray crystallographic study of one of them. Electrochemical oxidation of these compounds, studied by cyclic voltammetry, revealed that their oxidation peak potentials were less positive for those compounds in which neighboring group participation was geometrically possible. Pulse radiolysis studies provided evidence for bond formation between the amide moiety and sulfur on one-electron oxidation in cases where the moieties are juxtaposed. Furthermore, molecular constraints in spiro analogues revealed that S-O bonds are formed on one-electron oxidation. DFT calculations suggest that isomeric sigma*(SO) radicals are formed in these systems.

Interactions of arenes and thioethers resulting in facilitated oxidation.

Synthesis of 6-endo and 6-exomethylthio-2-endoarylbicyclo[2.2.1]heptanes was accomplished stereoselectively. The ionization energies, determined by photoelectron spectroscopy, and electrochemical oxidation potentials, determined by cyclic voltammetry, were lower for the 6-endomethylthio compounds than for their 6-exomethylthio analogues. Calculations supported the notion that facilitation of electron transfer in the 6-endomethylthio compounds results from through-space S...pi interaction.

In vivo biosensors.

Detailed understanding of the dynamics of living systems requires a means to monitor, in real time, changes in the levels of key components in response to specific stimuli. Applications involving cultured cells, tissue slices and implantation in living systems are discussed.