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In vitro microfluidic systems are increasingly used as an alternative to standard Petri dishes in bioengineering and metabolomic investigations, as they are expected to provide cellular environments close to the in vivo conditions. In this work, we combined the recently developed “metabolomics-on-a-chip” approach with metabolic flux analysis to model the metabolic network of the hepatoma HepG2/C3A cell line and to infer the distribution of intracellular metabolic fluxes in standard Petri dishes and microfluidic biochips. A high pyruvate reduction to lactate was observed in both systems, suggesting that the cells operate in oxygen-limited environments. Our results also indicate that HepG2/C3A cells in the biochip are characterized by a higher consumption rate of oxygen, presumably due to a higher oxygenation rate in the microfluidic environment. This leads to a higher entry of the ultimate glycolytic product, acetyl-CoA, into the Krebs cycle. These findings are supported by the transcriptional activity of HepG2/C3A cells in both systems since we observed that genes regulated by a HIF-1 (hypoxia-regulated factor-1) transcriptional factor were over expressed under the Petri conditions, but to a lesser extent in the biochip.

In this report, we experimentally demonstrate that improving the cis -regulatory region of a target promoter can significantly enhance the response to an otherwise poor inducer. The transcriptional factor (TF) BenR of Pseudomonas putida KT2440 is a member of the AraC/XylS family that activates the Pb promoter in response to benzoate. This TF can also trigger Pb activity in response to 3-methylbenzoate (3MBz), but with lower efficiency. Unlike other family members, BenR appears to recognize one operator partially overlapping the ?35 promoter region and which is followed by an upstream DNA sequence that lacks an essential motif for TF binding. By generating a promoter variant composed of two complete operator sequences, we observed an enhancement in the sensitivity of Pb to the two inducers. This effect was more pronounced in the case of 3MBz, for which the transcriptional response was approximately 4–5 times higher with the variant than with the wild type promoter. By comparing the responses of the promoters to different concentrations of the inducers, we observed that the modification of the BenR binding region changes the inherent logic of Pb from an amplifier-like behaviour, in which benzoate acts as the sole input, towards OR-gate behaviour, in which 3MBz acts as a second input. Using a mathematical model, we deduced that the second binding site engineered in the Pb promoter enhances the activity of BenR that is bound to the natural operator region, increasing the inducing sensitivity. This work demonstrates how the promiscuity or specificity of inducer recognition can be tuned in a regulatory network without TF mutation and suggests new strategies for the engineering of logic operations in living systems.

A novel functional iodothyronine analogue, TRC150094, which has a much lower potency toward thyroid hormone receptor (α1/β1) activation than triiodothyronine , has been shown to be effective at reducing adiposity in rats simultaneously receiving a high-fat diet (HFD). Here, by combining metabolic, functional and proteomic analysis, we studied how the hepatic and skeletal muscle phenotypes might respond to TRC150094 treatment in HFD-fed overweight rats. Drug treatment increased both the liver and skeletal muscle mitochondrial oxidative capacities without altering mitochondrial efficiency. Coherently, in terms of individual respiratory in-gel activity, blue-native analysis revealed an increased activity of complex V in the liver and of complexes II and V in tibialis muscle in TCR150094-treated animals. Subsequently, the identification of differentially expressed proteins and the analysis of their interrelations gave an integrated view of the phenotypic/metabolic adaptations occurring in the liver and muscle proteomes during drug treatment. TRC150094 significantly altered the expression of several proteins involved in key liver metabolic pathways, including amino acid and nitrogen metabolism, and fructose and mannose metabolism. The canonical pathways most strongly influenced by TRC150094 in tibialis muscle included glycolysis and gluconeogenesis, amino acid , fructose and mannose metabolism, and cell signaling. The phenotypic/metabolic influence of TRC150094 on the liver and skeletal muscle of HFD-fed overweight rats suggests the potential clinical application of this iodothyronine analogue in ameliorating metabolic risk parameters altered by diet regimens.

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RNA-binding proteins (RBPs) have essential roles in post-transcriptional regulation of gene expression. They bind sequence elements in specific mRNAs and control their splicing, transport, localization, translation, and stability. A complete understanding of RBP function requires identification of the target RNAs that an RBP regulates, the mechanisms by which the RBP regulates these targets, and the biological consequences for the cell in which these transactions occur. Antibodies are key tools in such studies: first, mRNA targets of RBPs can be identified by co-immunoprecipitation of RBPs with their associated RNAs followed by microarray analysis or sequencing; second, partner proteins can be identified by immunoprecipitation of the RBP followed by mass spectrometry ; third, the mechanisms and functions of RBPs can be inferred from loss-of-function studies employing antibodies that block RBP-RNA interactions. One potentially powerful approach to making antibodies for such studies is the generation of synthetic antibodies using phage display, which involves in vitro selection using a human-designed antibody library to generate antibodies that recognize a target protein . Using two well-characterized Drosophila RNA-binding proteins , Staufen and Smaug, for proof-of-principle, we demonstrate that synthetic antibodies can be generated and used either to perform RNA-coimmunoprecipitations (RIPs) to identify RBP-bound mRNAs, or to block RBP-RNA interactions. Given that synthetic antibody selection protocols are amenable to high-throughput antibody production, these results demonstrate that synthetic antibodies can be powerful tools for genome-wide studies of RBP function.

Climatic change is an increasing challenge for agriculture that is driving the development of suitable crops in order to ensure supply for both human nutrition and animal feed. In this context, it is increasingly important to understand the biochemical responses of cells to environmental cues at the whole system level, an aim that is being brought closer by advances in high throughput, cost-efficient plant metabolomics. To support molecular breeding activities, we have assessed the economic, technical and statistical feasibility of using direct mass spectrometry methods to evaluate the physiological state of maize ( Zea mays L.) plants grown under different stress conditions.

Fast cancer diagnosis represents a real necessity in applied medicine due to the importance of this disease. Thus, theoretical models can help as prediction tools. Graph theory representation is one option because it permits us to numerically describe any real system such as the protein macromolecules by transforming real properties into molecular graph topological indices. This study proposes a new classification model for proteins linked with human colon cancer by using spiral graph topological indices of protein amino acid sequences. The best quantitative structure–disease relationship model is based on eleven Shannon entropy indices. It was obtained with the Na?ve Bayes method and shows excellent predictive ability (90.92%) for new proteins linked with this type of cancer. The statistical analysis confirms that this model allows diagnosing the absence of human colon cancer obtaining an area under receiver operating characteristic of 0.91. The methodology presented can be used for any type of sequential information such as any protein and nucleic acid sequence.

Self-assembled peptide -based nanostructures have been the focus of research in the past decade because of their potential applications in various biological systems. Normally, small self-assembled peptide nanostructures contain hydrophobic moieties, therefore, their solubility in aqueous systems poses the important challenge in the field of molecular self-assembly in order to make effective use of these in a wide variety of applications. To improve their aqueous solubility, the self-assembled amphiphilic α,β-dehydrophenylalanine containing small glyco-dehydropeptides, Boc-Phe-ΔPhe-εAhx-GA ( I ) and H-Phe-ΔPhe-εAhx-GA ( II ) with glucosamine (GA) attached at the C-terminal through a 6-aminocaproic acid linker, were synthesized, demonstrating the formation of nanostructures in aqueous media, which were characterized by DLS , AFM and TEM . Further, nanostructure II reduced auric chloride to gold nanoparticles and formed a peptide –gold conjugate ( VII ). The feasibility of using the nanostructures I and II as nanovectors for drug delivery was demonstrated by loading hydrophobic molecules, eosin and N -fluoresceinyl-2-aminoethanol (FAE) dyes . Besides, these peptides displayed antimicrobial activity against Micrococcus flavus , Bacillus subtilis and Pseudomonas aeruginosa . All these results advocate the potential of these nanostructures as efficient vectors for drug delivery applications.

A graphical abstract is available for this content

A graphical abstract is available for this content