Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2. Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus). Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. (C) 2008 Elsevier B.V. All rights reserved.

Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology.

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the P(luxI) promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of "hidden interactions" on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.

High confidence prediction of essential genes in burkholderia cenocepacia

Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex.

Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae

Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.

Density-Profile Processes Describing Biological Signaling Networks: Almost Sure Convergence to Deterministic Trajectories

We introduce jump processes in R(k), called density-profile processes, to model biological signaling networks. Our modeling setup describes the macroscopic evolution of a finite-size spin-flip model with k types of spins with arbitrary number of internal states interacting through a non-reversible stochastic dynamics. We are mostly interested on the multi-dimensional empirical-magnetization vector in the thermodynamic limit, and prove that, within arbitrary finite time-intervals, its path converges almost surely to a deterministic trajectory determined by a first-order (non-linear) differential equation with explicit bounds on the distance between the stochastic and deterministic trajectories. As parameters of the spin-flip dynamics change, the associated dynamical system may go through bifurcations, associated to phase transitions in the statistical mechanical setting. We present a simple example of spin-flip stochastic model, associated to a synthetic biology model known as repressilator, which leads to a dynamical system with Hopf and pitchfork bifurcations. Depending on the parameter values, the magnetization random path can either converge to a unique stable fixed point, converge to one of a pair of stable fixed points, or asymptotically evolve close to a deterministic orbit in Rk. We also discuss a simple signaling pathway related to cancer research, called p53 module.

Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [³⁵S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

Vasodilatory effects of homologous adrenomedullin 2 and adrenomedullin 5 on isolated blood vessels of two species of eel

In mammals, adrenomedullin (AM) is a potent vasodilator through signalling pathways that involve the endothelium. In teleost fishes, a family of five AMs are present (AM1/4, AM2/3 and AM5) with four homologous AMs (AM1, AM2/3 and AM5) recently cloned from the Japanese eel, Anguilla japonica. Both AM2 and AM5 have been shown to be strong in vivo vasodepressors in eel, but the mechanism of action of homologous AMs on isolated blood vessels has not been examined in teleost fish. In this study, both eel AM2 and AM5 caused a marked vasodilation of the dorsal aorta. However, only AM5 consistently dilated the small gonadal artery in contrast to AM2 that had no effect in most preparations. Neither AM2 nor AM5 had any effect when applied to the first afferent branchial artery; in contrast, eel ANP always caused a large vasodilation of the branchial artery. In the dorsal aorta, indomethacin significantly reduced the AM2 vasodilation, but had no effect on the AM5 vasodilation. In contrast, removal of the endothelium significantly enhanced the AM5 vasodilation only. In the gonadal artery, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) significantly reduced the AM5 vasodilation suggesting a role for soluble guanylyl cyclase in the dilation, but l-NNA and removal of the endothelium had no effect. The results of this study indicate that AM2 and AM5 have distinct vasodilatory effects that may be due to the peptides signalling via different receptors to regulate vascular tone in eel.

Abordagens bio-inspiradas aplicadas ao estudo da cognição: um encontro entre biologia, psicologia e filosofia

Pós-graduação em Filosofia - FFC

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

Interfaccia grafica per l'elaborazione di immagini microscopiche di colture batteriche geneticamente modificate

Le moderne tecniche di imaging e i recenti sviluppi nel campo della visione computazionale consentono sempre più diffusamente l'utilizzo di metodi di image analysis, specialmente in ambito medico e biologico, permettendo un maggiore supporto sia alla diagnosi, sia alla ricerca. Il lavoro svolto in questa tesi si pone in un contesto di ricerca di carattere interdisciplinare, e riguarda il progetto e la realizzazione di un‘interfaccia grafica per l'analisi di colture batteriche geneticamente modificate, marcate con proteine fluorescenti (GFP), acquisite tramite un microscopio ad epifluorescenza. Nota la funzione di risposta del sistema di acquisizione delle immagini, l'analisi quantitativa delle colture batteriche è effettuata mediante la misurazione di proprietà legate all'intensità della risposta al marcatore fluorescente. L'interfaccia consente un'analisi sia globale dei batteri individuati nell'immagine, sia di singoli gruppi di batteri selezionati dall'utente, fornendo utili informazioni statistiche, sia in forma grafica che numerica. Per la realizzazione dell'interfaccia sono state adottate tecniche di ingegneria del software, con particolare enfasi alla interazione uomo-macchina e seguendo criteri di usability, al fine di consentire un corretto utilizzo dello strumento anche da parte di personale senza conoscenza in campo informatico.

Recombinant expression of molluscan hemocyanin (KLH) substructures in a prokaryotic system: E. coli

The recombinant expression of 19 different substructures of KLH in the prokaryotic sys-tem E. coli has been successfully achieved: each one of the eight single FUs a to h of both isoforms, KLH1 and KLH2, two substructures consisting of two consecutive FUs (KLH1-bc and KLH1-gh) as well as a cDNA encompassing KLH1-abc. All recombinant proteins, fused to an N-terminal 6xHis tag, have successfully been detected by immuno precipitation using monoclonal α-His-antibodies and polyclonal α-KLH1- and α-KLH2-antibodies. One exception remained: SP-KLH2-a, which was not detected by the α-His-antibodies. This allows speculations as to whether the coexpressed signal peptide can lead, at one hand, to the secretion of the recombinant protein, and on the other to the simultaneous cut-off of the leader peptide, which results in the splitting off of even more N-terminal 6xHis tag, leading to failed recognition by the appropriate antibodies. The comparison of native KLH with recombinantly expressed prokaryotic (E. coli) and eukaryotic (Sf9 insect cells) KLH was done using FU-1h. The weak detection by the polyclonal α-KLH1-antibodies of both recombinantly expressed proteins showed that the native protein was the best recognized. For the prokaryotic one, both the denaturation applied for solubilisation of the bacterial inclusion bodies and the inability of bacterial cells to add N-linked glycosylation, are the reason for the poor hybridization. In contrast, KLH1-h expressed in eukaryotic insect cells is likely to be glycosylated. The incubation with the α-KLH1-antibodies resulting in the same weak detection, however, revealed that the linked carbohydrate side chains are not those expected. The establishment of SOE-PCR, together with further improvement, has enabled the generation of a clone encompassing the complete subunit KLH1-abcdefgh. The se-quence analysis compared to the original KLH1 sequence showed, however, that the resulting recombinant protein is defective in two histidines, required for the copper bind-ing sites in FU-1b and FU-1d and in three disulfide bridges (FU-1a, FU-1b and FU 1g). This is due to polymerase-related nucleotide exchanges, resulting in a changed amino acid sequence. Nevertheless, all eight potential N-glycosylation sites are present, leading to the speculation that the recombinant protein can in theory be fully glycosylated, which is the most important aspect for the clinical applicability of recombinant KLH as an im-munotherapeutic agent. The improvement of this method elaborated during the present work indicates bright prospects for the future generation of a correct cDNA sequence encoding for the complete KLH2 subunit.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.