21 research outputs found

    Gastric-targeted drug delivery by learning from Helicobacter pylori

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    Drugs which would preferentially be absorbed through the stomach or upper small intestine are characterised by a narrow absorption window, due to their rapid transit time through these regions of the gastrointestinal tract and the insufficient contact time with their site of absorption. In order to address the problem of low bioavailability, which is related to the narrow absorption window, the development of drug delivery systems which adhere to the gastric epithelium and retain the drug in the stomach was an appealing approach. The present study suggests the exploitation of adhesins from Helicobacter pylori, in order to generate gastric-targeted drug delivery carriers, which mimic the bacterial mechanism and adhere to the gastric epithelium. In the present work, four different H. pylori adhesins were recombinantly expressed at a range of yields in the periplasmic space of Escherichia coli; the highest yield was observed for LabA. Although it did not influence the expression yield, the presence of a C terminal hexalysine tag in the expression construct of LabA enhanced the protein’s thermal stability and aqueous solubility, without affecting its secondary structure. The enhancement of solubility imposed obstacles to protein crystallisation; redesign of the expression construct led to the acquisition of protein crystals and a crystal structure, which was the biggest achievement of this work, as the crystal structure of LabA J99 has not been published. Although the crystal structure was determined, no specific ligands were identified for LabA J99. Consequently, recombinant BabA with known affinity for Leb was used to decorate model gastric targeted microparticles. After confirming the binding affinity of the BabA conjugated microparticles for Leb, polymer coating of the surface of the microparticles was examined for its protective effect against pepsin digestion of BabA, which constitutes the main challenge for the viability of the microparticles in vivo

    Experimental study of cadmium bioaccumulation in three Mediterranean marine bivalve species: correlation with selected biomarkers

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    An ecotoxicological study is presented, in which three marine bivalve species (Mytilus galloprovincialis, Callista chione, and Venus verrucosa) living in different habitats were studied for Cd bioaccumulation, under laboratory conditions. The bivalves, originating from a relatively polluted marine area of Greece (Saronicos Gulf), were exposed to 0.5 mg Cd L–1 seawater (4.4 mol Cd L–1 seawater) for 5, 10, 15, and 20 days. Control animals were kept in metal-free seawater as well. Three or four different parts of the organisms (gills, mantle, body, digestive system) were examined for the bioaccumulation of Cd, as well as the levels of three biomarkers (metallothioneins, acetylcholinesterase, lipid peroxidation). A depuration experiment was also carried out. During the experiment, the initial levels of Cd in the control animal tissues either decreased or remained constant and low. The organisms exhibited different behavior regarding Cd bioconcentration and biomarker responses as well as tissue distribution of Cd. After the depuration period, significant amounts of Cd remained in the organisms’ tissues, much higher than the respective levels in control animals

    The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

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    Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics

    Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori

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    Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation

    Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori

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    Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding

    Mechanistic investigations into the encapsulation and release of small molecules and proteins from a supramolecular nucleoside gel in vitro and in vivo

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    Supramolecular gels have recently emerged as promising biomaterials for the delivery of a wide range of bioactive molecules, from small hydrophobic drugs to large biomolecules such as proteins. Although it has been demonstrated that each encapsulated molecule has a different release profile from the hydrogel, so far diffusion and steric impediment have been identified as the only mechanisms for the release of molecules from supramolecular gels. Erosion of a supramolecular gel has not yet been reported to contribute to the release profiles of encapsulated molecules. Here, we use a novel nucleoside-based supramolecular gel as a drug delivery system for proteins with different properties and a hydrophobic dye and describe for the first time how these materials interact, encapsulate and eventually release bioactive molecules through an erosion-based process. Through fluorescence microscopy and spectroscopy as well as Small Angle X-ray scattering, we show that the encapsulated molecules directly interact with the hydrogel fibres - rather than being physically entrapped in the gel network. The ability of these materials to protect proteins against enzymatic degradation is also demonstrated here for the first time. In addition, the released proteins were proven to be functional in vitro. Real-time fluorescence microscopy together with macroscopic release studies confirm that erosion is the key release mechanism. In vivo, the gel completely degrades after two weeks and no signs of inflammation are detected, demonstrating its in vivo safety. By establishing the contribution of erosion as a key driving force behind the release of bioactive molecules from supramolecular gels, this work provides mechanistic insight into the way molecules with different properties are encapsulated and released from a nucleoside-based supramolecular gel and sets the basis for the design of more tailored supramolecular gels for drug delivery applications

    Gastric-targeted drug delivery by learning from Helicobacter pylori

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    Drugs which would preferentially be absorbed through the stomach or upper small intestine are characterised by a narrow absorption window, due to their rapid transit time through these regions of the gastrointestinal tract and the insufficient contact time with their site of absorption. In order to address the problem of low bioavailability, which is related to the narrow absorption window, the development of drug delivery systems which adhere to the gastric epithelium and retain the drug in the stomach was an appealing approach. The present study suggests the exploitation of adhesins from Helicobacter pylori, in order to generate gastric-targeted drug delivery carriers, which mimic the bacterial mechanism and adhere to the gastric epithelium. In the present work, four different H. pylori adhesins were recombinantly expressed at a range of yields in the periplasmic space of Escherichia coli; the highest yield was observed for LabA. Although it did not influence the expression yield, the presence of a C terminal hexalysine tag in the expression construct of LabA enhanced the protein’s thermal stability and aqueous solubility, without affecting its secondary structure. The enhancement of solubility imposed obstacles to protein crystallisation; redesign of the expression construct led to the acquisition of protein crystals and a crystal structure, which was the biggest achievement of this work, as the crystal structure of LabA J99 has not been published. Although the crystal structure was determined, no specific ligands were identified for LabA J99. Consequently, recombinant BabA with known affinity for Leb was used to decorate model gastric targeted microparticles. After confirming the binding affinity of the BabA conjugated microparticles for Leb, polymer coating of the surface of the microparticles was examined for its protective effect against pepsin digestion of BabA, which constitutes the main challenge for the viability of the microparticles in vivo

    Trace metals distribution and chemical behaviour in a coastal marine area affected by industrial pollution CNW Saronicos Gulf

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    The main objectives of this thesis were: α) to investigate the levels and behaviour of heavy metals in the marine environment of the NW Saronikos gulf, where industrial activity, coastal towns and a unique geological background (NW edge of the Hellenic volcanic arc) are combined and b) to assess the impact of the coastal industries emphasizing on the oil refinery ‘’MOTOR OIL HELLAS’’. After extended bibliographic search 8 seasonal samplings were conducted (2006-2007) in 16 stations of various depths using equipment of the laboratory of environmental chemistry and boats supplied mainly by ‘’MOTOR OIL HELLAS’’. The heavy metals Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, V, Zn were determined in seawater, sediment and processed effluent samples using standard and published methods that included various pre-concentration techniques and final measurement with atomic absorption spectrometry. The methods were validated in order to estimate the limits of detection, repeatability and accuracy. The data of the present study were compared with similar data from other parts of Saronikos gulf and other marine areas. Finally the data were recorded in a specifically designed access database in order to be readily accessible for future comparisons. Despite the fact that near the industries the coasts appear aesthetically deteriorated the marine environment is not severely influenced by heavy metal pollution due to adequate water circulation and dilution. Therefore it is possible to achieve restoration of the coasts in combination with the establishment of regular monitoring to prevent environmental pollution.Οι κύριοι στόχοι της διατριβής ήταν: α) να διερευνηθούν τα επίπεδα και η συμπεριφορά των βαρέων μέταλλων στο θαλάσσιο περιβάλλον του ΒΔ Σαρωνικού, όπου συνδυάζονται η βιομηχανική επιβάρυνση, παράκτιοι οικισμοί και ιδιαίτερο γεωλογικό υπόβαθρο (βδ άκρο του ελληνικού ηφαιστειακού τόξου) και β) να εκτιμηθεί η επίδραση των παρακτίων βιομηχανιών με έμφαση στο διυλιστήριο της MOTOR OIL HELLAS. Έγινε εκτεταμένη βιβλιογραφική έρευνα, και πραγματοποιήθηκαν 8 εποχιακές δειγματοληψίες (2006-2007) σε 16 σταθμούς σε διάφορα βάθη με εξοπλισμό πεδίου του Εργ. Χημείας Περιβάλλοντος και πλωτά μέσα κυρίως από τη MOTOR OIL. Στα δείγματα θαλασσινού νερού, ιζημάτων και επεξεργασμένων απόβλητων προσδιορίστηκαν τα βαρέα μέταλλα Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, V, Zn με πρότυπες και δημοσιευμένες μεθόδους που περιλάμβαναν διάφορα σταδία προκατεργασίας και τελική μέτρηση με Φασματόμετρα Ατομικής Απορρόφησης. πραγματοποιήθηκε επικύρωση των μεθόδων για την εκτίμηση του ορίου ανίχνευσης, της επαναληψιμότητας και ορθοτητας. τα δεδομενα διερευνηθηκαν στατιστικα και έγινε σύγκριση με αντίστοιχα δεδομένα από τον Σαρωνικό κόλπο και άλλες θαλάσσιες περιοχές. τέλος τα δεδομένα καταχωρήθηκαν σε ειδικά σχεδιασμένη βάση ACCESS, ώστε να είναι διαθέσιμα για μελλοντικές συγκρίσεις. παρά την κακή αισθητική εικόνα των ακτών δίπλα στις βιομηχανικές μονάδες το θαλάσσιο περιβάλλον της περιοχής μελέτης δεν παρουσιάζει ιδιαίτερη επιβάρυνση ως προς τα βαρέα μέταλλα λόγω της καλής θαλάσσιας κυκλοφορίας. άρα είναι εφικτό να σχεδιαστεί ανάπλαση και εξυγίανση και θα πρέπει να συνδυαστεί με καθιέρωση τακτικού περιβαλλοντικού έλεγχου

    Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression

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    Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein’s pI is suggested

    A Field Study of Age Discrimination in the Workplace: The Importance of Gender and Race. Pay the Gap

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    Purpose: The study examines whether age intersects with gender and race during the initial stage of the hiring process and affects access to vacancies outcomes and wage sorting. Methodology: In order to answer the research question the study collects data from four simultaneous field experiments in England. The study compares the labour market outcomes of younger White British men with those of older White British men and women, and with those of older Black British men and women. The study concentrates on low-skilled vacancies in hospitality and sales in the private sector. Findings: The results of this study indicate that older White British men and women, as well as older Black British men and women, experience occupational access constraints and are sorted into lower-paid jobs than younger White British men. The level of age discrimination is found to be higher for Black British men and women. In addition, Black British women experience the highest level of age discrimination. These patterns may well be in-line with prejudices against racial minority groups and stereotypical sexist beliefs that the physical strengths and job performance of women decline earlier than they do for men. Originality/value: This research presents for the first-time comparisons of access to vacancies and wage sorting between younger male racial majorities and older male racial majorities, older female racial majorities, older male racial minorities, and older female racial minorities. In addition, the driven mechanism of the assigned differences is explored. Because the study has attempted to minimise the negative employer stereotypes vis-à-vis older employees, with respect to their motivation, productivity, and health, such prejudices against older individuals may be considered Taste-based discrimination. Implications: If prejudices against older individuals are present, then anti-discrimination legislation may be the appropriate response, especially for racial minorities and women. Eliminating age discrimination in selection requires firms to adopt inclusive HR policies at the earliest stages of the recruitment process
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