Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates

A preliminary investigation into the use of alginates for the lifting and enhancement of fingermarks in blood

Recent studies have reported the use of alginate in the lifting and subsequent enhancement of footwear marks in blood. A study was set up to assess the use of such a method in the treatment of fingermarks in blood on a variety of porous, non-porous and semi-porous surfaces. Other variables included ageing of the fingermarks in blood and the application of chemicals prior to or post-alginate lifting. All different variations were compared to direct chemical treatment of the substrate. The results demonstrated that alginate is not compatible with certain substrates (e.g. glass and tile). On substrates that were compatible with alginate (e.g. fabric and paper), the enhanced fingermarks on the alginate cast and the enhanced fingermarks on the post-alginate substrates appeared, overall, inferior compared to direct chemical enhancement without the use of alginate. A further variation using water-based protein stains directly mixed with the alginate appeared to provide enhancement directly on the substrate as well as simultaneous lifting and enhancing the fingermarks in blood on the alginate cast

Alginate Scaffolds for Mesenchymal Stem Cell Cardiac Therapy: Influence of Alginate Composition

Despite the success of alginate scaffolds and mesenchymal stem cells (MSCs) therapy in cardiac failure treatment, the impact of the physicochemical environment provided by alginate matrices on cell behavior has never been investigated. The purpose of this work was double: to determine the alginate composition influence on (1) encapsulated rat MSC viability, paracrine activity, and phenotype in vitro and (2) cardiac implantability and in vivo biocompatibility of patch shape scaffolds. Two alginates, differing in composition and thus presenting different mechanical properties when hydrogels, were characterized. In both cases, encapsulated MSC viability was maintained at around 75%, and their secretion characteristics were retained 28 days postencapsulation. In vivo study revealed a high cardiac compatibility of the tested alginates: cardiac parameters were maintained, and rats did not present any sign of infection. Moreover, explanted hydrogels appeared surrounded by a vascularized tissue. However, scaffold implantability was highly dependent on alginate composition. G-type alginate patches, presenting higher elastic and Young moduli than M-type alginate patches, showed a better implantation easiness and were the only ones that maintained their shape and morphology in vivo. As a consequence of alginate chemical composition and resulting hydrogel structuration, G-type alginate hydrogels appear to be more adapted for cardiac implantation

Comparative study of encapsulated rhizome extract of Alpinia purpurata (Zingeberaceae) in alginate and alginate-chitosan

Encapsulation is a coating process of bioactive compound. Alpinia purpurata has been well known as lengkuas merah an Asian tropical herbal which contain phenylpropanoid, phenolic and flavonoid. Phenolic and flavonoid compounds is an agent that can be used as anti cancer. This research aim is to create a product of Alpinia purpurata extract which encapsulated in alginate or alginate-chitosan. Theproduct of encapsulated has been observed towards SEM ( Scanning Electron Microscophy) and spectrocophy Infra-Red method. Encapsulated product of lengkuas merah extract made through extrusion method in alginate and chitosan with ratio 1:1 (w/w) then dripped in 2% CaCl2.The Alpinia purpurata/alginate/chitosan microcapsules (APCAM) is better than alginate microcapsules (APAM)

Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells

Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n=15) were given a test meal of 200g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p=0.003). Sub-group B (n=9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p=0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p=0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p=0.009) and 35% (p=0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p<0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification

A note on the effect of calcium alginate coating on quality of refrigerated strawberries.

peer-reviewedAn alginate-based edible coating was investigated for the preservation of the quality of strawberries during cold storage (5 °C). Strawberries were immersed, successively, in sodium alginate and calcium chloride solutions to generate a surface coating of calcium alginate. The quality of coated and non-coated strawberries was evaluated by weight loss, visible decay, titratable acidity, total soluble solids and reducing sugar concentration over a 14-day storage period. Results showed that coating with calcium alginate had no significant effects on weight loss or physicochemical parameters when compared to control fruit, but it did result in the postponement of visible decay during refrigerated storage

Alginate-containing Compositions For Use In Battery Applications

A silicon-based anode comprises an alginate-containing binder. The many carboxy groups of alginate bind to a surface of silicon, creating strong, rigid hydrogen bonds that withstand battery cycling. The alginate-containing binder provides good performance to the anode by (1) improving the capacity of the anode in comparison to other commercially-available binders, (2) improving Columbonic efficiency during charging and discharging cycles, and (3) improving stability during charging and discharging cycles.Clemson UniversityGeorgia Tech Research Corporatio

Recombinant protein immobilisation and display by alginate : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.

Biopolymers are a diverse group of organic materials with important applications in a number of industries. Their ability to adsorb and encapsulate compounds has been widely utilised in both biotechnologies and pharmaceuticals. In the last decade, biopolymers have been given new and enhanced functionality, including the separation and purification of compounds. This field is of increasing relevance as advances in the bacterial cell culture process have improved productivity in the biomanufacturing industry, with the establishment of several bacterial host cell lines and optimised protein production systems. This increase in upstream productivity is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. While previous studies have generated functionalised protein biopolymers using polyhydroxyalkanoate (PHA) biopolyester beads, very few studies have examined the commercially significant biopolymer alginate. Alginate is an exopolysaccharide produced by algae and some bacteria, and is widely utilised in food, pharmaceutical, and biomedical industries because of its stabilising, haemostatic, biocompatible properties and its modifiable structure. In this study, a partially functional alginate-binding recombinant protein was produced, which contained an α-amylase domain from Bacillus licheniformis (BLA) translationally fused to the alginate-binding domain of Pseudomonas aeruginosa AlgX – an alginate acetyltransferase. An Ssp DnaB mini-intein was included between BLA and AlgX to facilitate recovery of BLA, following immobilisation and display on the surface of alginate. However, aberrant activity of the intein caused total cleavage of the recombinant protein between its BLA and AlgX domains before it could be recovered from the protein production system. Additionally, the absence of a key cysteine residue in the alginate-binding domain prevented the formation of a disulfide bond, which is an essential structural element for the folding and functionality of this region. While this study was unable to overcome intein hyperactivity, functional analysis of the BLA domain showed consistent and significant levels of α-amylase activity, leading to a positive outlook for the functionality of a full-length recombinant protein if proper intein activity can be restored and the necessary cysteine included. In this way, alginate could be specifically functionalised with a desired protein, and in turn, alginate beads could be used for the separation and enrichment of target proteins

Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens.

Advances in nanotechnology have demonstrated potential application of nanoparticles (NPs) for effective and targeted drug delivery. Here we investigated the antimicrobial and immunological properties and the feasibility of using NPs to deliver antimicrobial agents to treat a cutaneous pathogen. NPs synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy (EM) imaging, chitosan-alginate NPs were found to induce the disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate NPs also exhibited anti-inflammatory properties as they inhibited P. acnes-induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide (BP), a commonly used antiacne drug, was effectively encapsulated in the chitosan-alginate NPs and demonstrated superior antimicrobial activity against P. acnes compared with BP alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate NP-encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components

Daily ingestion of alginate reduces energy intake in free-living subjects

Sodium alginate is a seaweed-derived fibre that has previously been shown to moderate appetite in models of acute feeding. The mechanisms underlying this effect may include slowed gastric clearance and attenuated uptake from the small intestine. In order to assess whether alginate could be effective as a means of appetite control in free-living adults, 68 males and females (BMI range: 18.50-32.81 kgl M-2) completed this randomised, controlled two-way crossover intervention to compare the effects of 7 day daily ingestion of a strong-gelling sodium alginate formulation against a control. A sodium alginate with a high-guluronate content was chosen because, upon ingestion, it forms a strong gel in the presence of calcium ions. Daily preprandial ingestion of the sodium alginate formulation produced a significant 134.8 kcal (7%) reduction in mean daily energy intake. This reduced energy intake was underwritten by significant reductions in mean daily carbohydrate, sugar, fat, saturated fat and protein intakes. The absence of any significant interaction effects between the main effect of preload type and those of gender, BMI classification and/or timing of preload delivery indicates the efficacy of this treatment for individuals in different settings. These findings suggest a possible role for a strong-gelling sodium alginate formulation in the future management of overweight and obesity. (C) 2008 Elsevier Ltd. All rights reserved