Enzyme and Cell Engineering - Molecular Recognition and Biocatalysis

MDAnalysis (http://mdanalysis.org) is a library for structural and temporal analysis of molecular dynamics (MD) simulation trajectories and individual protein structures.MD simulations of biological molecules have become an important tool to elucidate the relationship between molecular structure and physiological function.Simulations are performed with highly optimized software packages on HPC resources but most codes generate output trajectories in their own formats so that the development of new trajectory analysis algorithms is confined to specific user communities and widespread adoption and further development is delayed.MDAnalysis addresses this problem by abstracting access to the raw simulation data and presenting a uniform object-oriented Python interface to the user.It thus enables users to rapidly write code that is portable and immediately usable in virtually all biomolecular simulation communities.The user interface and modular design work equally well in complex scripted work flows, as foundations for other packages, and for interactive and rapid prototyping work in IPython / Jupyter notebooks, especially together with molecular visualization provided by nglview and time series analysis with pandas.MDAnalysis is written in Python and Cython and uses NumPy arrays for easy interoperability with the wider scientific Python ecosystem.It is widely used and forms the foundation for more specialized biomolecular simulation tools.MDAnalysis is available under the GNU General Public License v2.

Many of the intricate pathways of apoptosis that instruct a cell to kill itself involve the convergence of key proteins on the membranes of mitochondria. Such proteins induce the permeabilization of mitochondrial membranes and the release of caspase enzymes and nuclease activators that set in motion the final stages of programmed cell death. Now, as Brenner and Kroemer discuss in their Perspective, a proapoptotic transcription factor called TR3 has been found to move from its normal location in the nucleus to the mitochondria and to promote release of cytochrome c, a key event in apoptosis (Li et al.)

The technique of molecular imprinting allows the formation of specific recognition sites in synthetic polymers through the use of templates or imprint molecules. These recognition sites mimic the binding sites of antibodies and other biological receptor molecules. Molecularly imprinted polymers can therefore be used in applications relying on specific molecular binding events. The stability, ease of preparation and low cost of these materials make them particularly attractive. This review focuses on recent developments and advances in the field of molecularly imprinted materials, with special emphasis on applications in immunoassays and sensors recently developed by our group and by others.

We demonstrate, on the example of trypsin, the use of water-soluble molecularly imprinted polymer microgels as specific enzyme inhibitors. Using a strong anchoring monomer, methacryloylaminobenzamidine, the growing polymer chains are confined to close proximity of the substrate recognition site of our model enzyme. The microgels bind selectively trypsin over other proteins of similar size and molecular weight, and show competitive inhibition of trypsin with an inhibition constant K(i) of 79 nM, making them more potent inhibitors than the low molecular-weight competitive inhibitor benzamidine by almost 3 orders of magnitude. We believe that these tailor-made materials with biological activity have potential for future drug development that extends beyond enzyme inhibition.

When studying biological membranes, Molecular Dynamics (MD) simulations reveal to be quite complementary to experimental techniques. Because the simulated systems keep increasing both in size and complexity, the analysis of MD trajectories need to be computationally efficient while being robust enough to perform analysis on membranes that may be curved or deformed due to their size and/or protein-lipid interactions. This work presents a new software named FATSLiM ('Fast Analysis Toolbox for Simulations of Lipid Membranes') that can extract physical properties from MD simulations of membranes (with or without interacting proteins). Because it relies on the calculation of local normals, FATSLiM does not depend of the bilayer morphology and thus can handle with the same accuracy vesicles for instance. Thanks to an efficiency-driven development, it is also fast and consumes a rather low amount of memory. AVAILABILITY AND IMPLEMENTATION: FATSLiM (http://fatslim.github.io) is a stand-alone software written in Python. Source code is released under the GNU GPLv3 and is freely available at https://github.com/FATSLiM/fatslim A complete online documentation including instructions for platform-independent installation is available at http://pythonhosted.org/fatslim CONTACT: sebastien.buchoux@u-picardie.frSupplementary information: Supplementary data are available at Bioinformatics online.

Surfactin is a lipoheptapeptide produced by several Bacillus species and identified for the first time in 1969. At first, the biosynthesis of this remarkable biosurfactant was described in this review. The peptide moiety of the surfactin is synthesized using huge multienzymatic proteins called NonRibosomal Peptide Synthetases. This mechanism is responsible for the peptide biodiversity of the members of the surfactin family. In addition, on the fatty acid side, fifteen different isoforms (from C12 to C17) can be incorporated so increasing the number of the surfactin-like biomolecules. The review also highlights the last development in metabolic modeling and engineering and in synthetic biology to direct surfactin biosynthesis but also to generate novel derivatives. This large set of different biomolecules leads to a broad spectrum of physico-chemical properties and biological activities. The last parts of the review summarized the numerous studies related to the production processes optimization as well as the approaches developed to increase the surfactin productivity of Bacillus cells taking into account the different steps of its biosynthesis from gene transcription to surfactin degradation in the culture medium.

The polyphenols extraction from grape seeds was studied. In order to increase the polyphenols yield and the extraction kinetics, three different pre-treatments were applied on grape seeds: pulsed electric fields (PEF) (8–20 kV/cm, 0–20 ms), high-voltage electrical discharges (HVED) (10 kA/40 kV, 1 ms) and grinding (180 W, 40 s). These electrical treatments can damage cell membranes (PEF/HVED) and cell walls (HVED) thus accelerating the compounds extraction but have different effects on the product fragmentation. The diffusion was then performed after pre-treatment with a mixture of water and ethanol. The PEF efficiency was improved when the treatment was performed at 50 °C in the presence of ethanol. PEF-assisted diffusion was then compared to HVED-assisted diffusion and to diffusion of ground grape seeds. These two pre-treatments increased both the extraction kinetics and the maximum polyphenols yield. For both cases, the final polyphenols content was 9 g GAE/100 g DM after 15 min of extraction with grinding and after 60 min with HVED. When PEF were applied in a suspension containing ethanol, the maximum polyphenols yield was also reached after 60 min. The subsequent solid–liquid separation was faster with PEF as compared to ground and HVED-treatments.

Bcl-2 is a prosurvival factor that reportedly prevents the nonspecific permeabilization of mitochondrial membranes, yet enhances specific ADP/ATP exchange by these organelles. Here, we show that Bcl-2 enhances the ADP/ATP exchange in proteoliposomes containing the purified adenine nucleotide translocase (ANT) in isolated mitochondria and mitoplasts, as well as in intact cells in which mitochondrial matrix ATP was monitored continuously using a specific luciferase-based assay system. Conversely, Bax, which displaces Bcl-2 from ANT in apoptotic cells, inhibits ADP/ATP exchange through a direct action on ANT. The Bax-mediated inhibition of ADP/ATP exchange can be separated from Bax-stimulated formation of nonspecific pores by ANT. Chemotherapy-induced apoptosis caused an inhibition of ANT activity, which preceded the loss of the mitochondrial transmembrane potential and could be prevented by overexpression of Bcl-2. These data are compatible with a model of mitochondrial apoptosis regulation in which ANT interacts with either Bax or Bcl-2, which both influence ANT function in opposing manners. Bcl-2 would maintain the translocase activity at high levels, whereas Bax would inhibit the translocase function of ANT.

Abstract A general protocol to synthesize superparamagnetic molecularly imprinted polymer particles, using a RAFT‐mediated approach, is described. S‐ propranolol‐imprinted composites were obtained by functionalizing commercially available amino‐modified Fe3O4 nanoparticles with a trithiocarbonate agent and subsequently by polymerizing thin molecularly imprinted layers. Different parameters were optimized and their effect on both nanomorphology and imprinting behaviour was studied. Optimum conditions allowed the synthesis of 40 nm composite particles with a 7 nm MIP shell, exhibiting superparamagnetic properties and specific molecular recognition of S‐ propranolol. The possibility of fine‐tuning the surface properties of the particles is demonstrated by using the “living” nature of active RAFT fragments present on the surface of the composites to further functionalize the particles with ethylene glycol methacrylate phosphate polymer brushes.

ABSTRACT: Human papillomavirus (HPV), an etiological agent of cervical cancer (CC), has infected humans since ancient times. Amerindians are the furthest migrants out of Africa, and they reached the Americas more than 14,000 years ago. Some groups still remain isolated, and some migrate to towns, forming a gradient spanning urbanization. We hypothesized that, by virtue of their history, lifestyle, and isolation from the global society, remote Amerindian women have lower HPV diversity than do urban women (Amerindian or mestizo). Here we determined the diversity of the 25 most relevant cervical HPV types in 82 Amerindians spanning urbanization (low, medium, and high, consistent with the exposure to urban lifestyles of the town of Puerto Ayacucho in the Venezuelan Amazonas State), and in 29 urban mestizos from the town. Cervical, anal, oral, and introitus samples were taken, and HPVs were typed using reverse DNA hybridization. A total of 23 HPV types were detected, including 11 oncogenic or high-risk types, most associated with CC. Cervical HPV prevalence was 75%, with no differences by group, but Amerindians from low and medium urbanization level had significantly lower HPV diversity than mestizos did. In Amerindians, but not in mestizos, infections by only high-risk HPVs were higher than coinfections or by exclusively low-risk HPVs. Cervical abnormalities only were observed in Amerindians (9/82), consistent with their high HPV infection. The lower cervical HPV diversity in more isolated Amerindians is consistent with their lower exposure to the global pool, and transculturation to urban lifestyles could have implications on HPV ecology, infection, and virulence.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

A new, robust technique for preparing highly selective molecularly imprinted films (see Figure) is presented. Spin‐coating is used to spread a pre‐polymerization mixture on a substrate, which is subsequently cured with UV light. Important aspects of this technique include the use of a novel polymeric porogen dissolved in a low‐volatility solvent to generate porous morphologies with enhanced binding capacities.

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.

The objective of this study was to investigate the effects of pulsed electric field (PEF) and high-voltage electrical discharges (HVED) application on the efficiency of aqueous extraction of total soluble matter and polyphenols from grape skins ( Vitis vinifera L.) at different temperatures within 20-60 degrees C. The highest level of polyphenol concentration C was reached after about 60 min of extraction for HVED treatment: C(HVED) = 21.4 +/- 0.8 micromol of gallic acid equivalent (GAE)/g of dry matter (DM). Almost the same level of C was reached after 180 min of extraction for the PEF-treated skins. These levels exceeded the value C = 19.1 +/- 0.5 micromol of GAE/g of DM for the untreated samples. The difference between degrees Brix values for HVED-treated and untreated systems decreased with temperature increase (from 40 to 60 degrees C), but a large difference in the total amount of polyphenols was observed for HVED-treated and untreated systems. The activation energies were W(u) = 31.3 +/- 3.7 kJ/mol and W(PEF) = 28.9 +/- 5.5 kJ/mol for untreated and PEF-treated systems, respectively.

A series of mono-, di- and tri-valent iminosugars based on oligoethylene scaffolds and N-substituted deoxynojirymicin epitopes have been synthesized by "click chemistry" to study the effect of multivalency on glycosidase inhibition. Biological evaluation evidenced differences in the inhibition trends as a function of the enzyme nature. The results demonstrate that multivalency can be used in some case to modulate both the affinity and the selectivity of glycosidase inhibition.

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy.

The electrochemical behavior of stainless steels (SS) in natural waters is characterized by the ennoblement of their free corrosion potential (E(corr)). This phenomenon depends strongly on the settlement of biofilms on SS surfaces. Many hypotheses have been proposed to explain the biofilm action, in particular the enzymatic catalysis plays an important role by shifting the cathodic and/or anodic processes. However, there are still only few studies relating the use of purified enzymes. In contrast with bacteria-associated corrosion, the direct influence of enzymes is still poorly documented. The aim of this review is to show the benefits of the enzymatic approach in the study of biocorrosion. Indeed, enzymatic systems may constitute convenient models to mimic microbial influenced corrosion and to evaluate the behavior of metallic materials in natural waters.

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

Chemical nanosensors with a submicrometer core–shell composite design, based on a polymer core, a molecularly imprinted polymer (MIP) shell for specific analyte recognition, and an interlayer of gold nanoparticles for signal amplification, are described. SERS measurements on single nanosensors yield detection limits of 10−7 M for the β-blocker propranolol, several orders of magnitude lower than on plain MIP spheres.

Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.