Bioactive Properties of Mushroom (Agaricus bisporus) Stipe Extracts

The antibacterial activity and total phenolic (TP) content of Agaricus bisporus stipes were assessed using solvent and water extracts to determine its bioactivity. Extraction methods included accelerated solvent extraction (ASE) and hot water followed by membrane concentration. Water extract from ASE had the highest TP of 1.08 gallic acid equivalents (GAE)/g dry weight (DW) followed by ethanol at 0.61 mg GAE/g DW and 0.11 mg GAE/g DW for acetone. Acetone extracts inhibited Escherichia coli and Staphylococcus aureus at less than 50%; ethanol inhibited E. coli at 61.9% and S. aureus at 56.6%; and ASE water inhibited E. coli at 78.6% and S. aureus at 65.4%. The TP content of membrane concentrated extract of mushroom was 17 mg GAE in 100 mL. Membrane concentrated water extracts had a higher percentage inhibition on S. aureus than E. coli. Overall, the results were promising for further application of mushroom stipe extracts as a functional food additive. Practical Applications Mushrooms are known for their health benefits and have been identified as a good source of nutrients. The highly perishable nature of mushrooms warrants further processing and preservation to minimize losses along the supply chain. This study explores the possibility of adding value to mushroom stipes, a by-product of the fresh mushroom industry. The extracts assessed indicate the antibacterial activity and phenolic content, and the potential of using these extracts as functional ingredients in the food industry. This study provides valuable information to the scientific community and to the industries developing novel ingredients to meet the market demand for natural food additives.

Bioactive Properties of Mushroom (Agaricus bisporus) Stipe Extracts

The antibacterial activity and total phenolic (TP) content of Agaricus bisporus stipes were assessed using solvent and water extracts to determine its bioactivity. Extraction methods included accelerated solvent extraction (ASE) and hot water followed by membrane concentration. Water extract from ASE had the highest TP of 1.08 gallic acid equivalents (GAE)/g dry weight (DW) followed by ethanol at 0.61 mg GAE/g DW and 0.11 mg GAE/g DW for acetone. Acetone extracts inhibited Escherichia coli and Staphylococcus aureus at less than 50%; ethanol inhibited E. coli at 61.9% and S. aureus at 56.6%; and ASE water inhibited E. coli at 78.6% and S. aureus at 65.4%. The TP content of membrane concentrated extract of mushroom was 17 mg GAE in 100 mL. Membrane concentrated water extracts had a higher percentage inhibition on S. aureus than E. coli. Overall, the results were promising for further application of mushroom stipe extracts as a functional food additive. Practical Applications Mushrooms are known for their health benefits and have been identified as a good source of nutrients. The highly perishable nature of mushrooms warrants further processing and preservation to minimize losses along the supply chain. This study explores the possibility of adding value to mushroom stipes, a by-product of the fresh mushroom industry. The extracts assessed indicate the antibacterial activity and phenolic content, and the potential of using these extracts as functional ingredients in the food industry. This study provides valuable information to the scientific community and to the industries developing novel ingredients to meet the market demand for natural food additives.

The Use of Australian Honey in Moist Wound Management

This project has investigated the correlation between antimicrobial activity and the non-peroxide activity of particular Leptospermum-derived honeys. Initial investigations tested the efficacy of these honeys against one particular bacteria (Staphylococcus aureus), while later studies assessed the effect of this honey on a range of food pathogens, animal pathogens (e.g. in mastitis) and human pathogens (e.g. golden Staph). Using honeys sourced from around Australia and screened against several reference microorganisms, the anti-microbial potency of one particular Australian honey (from Leptospermum polygalifolium) was shown to be comparable to NZ Manuka honey (from L.scoparium). The results of this screening of active honeys against pathogenic bacteria has supported the registration of honey by Capilano Honey Limited as a “Drug” with the Therapeutic Goods Administration based on its antimicrobial activity.

Primary structure of a Thomsen-Friedenreich-antigen-specific lectin, jacalin [Artocarpus integrifolia (jack fruit) agglutinin]. Evidence for the presence of an internal repeat

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

3D QSAR studies of N-4-arylacryloylpiperazin-1-yl-phenyl-oxazolidinones: A novel class of antibacterial agents

Three-dimensional QSAR studies for N-4-arylacryloylpiperazin-1-yl-phenyl-oxazolidinones were conducted using TSAR 3.3. The in vitro activities (MICs) of the compounds against Staphylococcus aureus ATCC 25923 exhibited a strong correlation with the prediction made by the model developed in the present study.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Researching effective approaches to cleaning in hospitals: Protocol of the REACH study, a multi-site stepped-wedge randomised trial

Background The Researching Effective Approaches to Cleaning in Hospitals (REACH) study will generate evidence about the effectiveness and cost-effectiveness of a novel cleaning initiative that aims to improve the environmental cleanliness of hospitals. The initiative is an environmental cleaning bundle, with five interdependent, evidence-based components (training, technique, product, audit and communication) implemented with environmental services staff to enhance hospital cleaning practices. Methods/design The REACH study will use a stepped-wedge randomised controlled design to test the study intervention, an environmental cleaning bundle, in 11 Australian hospitals. All trial hospitals will receive the intervention and act as their own control, with analysis undertaken of the change within each hospital based on data collected in the control and intervention periods. Each site will be randomised to one of the 11 intervention timings with staggered commencement dates in 2016 and an intervention period between 20 and 50 weeks. All sites complete the trial at the same time in 2017. The inclusion criteria allow for a purposive sample of both public and private hospitals that have higher-risk patient populations for healthcare-associated infections (HAIs). The primary outcome (objective one) is the monthly number of Staphylococcus aureus bacteraemias (SABs), Clostridium difficile infections (CDIs) and vancomycin resistant enterococci (VRE) infections, per 10,000 bed days. Secondary outcomes for objective one include the thoroughness of hospital cleaning assessed using fluorescent marker technology, the bio-burden of frequent touch surfaces post cleaning and changes in staff knowledge and attitudes about environmental cleaning. A cost-effectiveness analysis will determine the second key outcome (objective two): the incremental cost-effectiveness ratio from implementation of the cleaning bundle. The study uses the integrated Promoting Action on Research Implementation in Health Services (iPARIHS) framework to support the tailored implementation of the environmental cleaning bundle in each hospital. Discussion Evidence from the REACH trial will contribute to future policy and practice guidelines about hospital environmental cleaning. It will be used by healthcare leaders and clinicians to inform decision-making and implementation of best-practice infection prevention strategies to reduce HAIs in hospitals. Trial registration Australia New Zealand Clinical Trial Registry ACTRN12615000325​505

Novel surgical and imaging methods of the middle ear and temporal bone

Välikorvaleikkauksiin usein liittyvän välikorvan ja kuuloluuketjun kirurgisen rekonstruktion tavoitteena on luoda olosuhteet, jotka mahdollistavat hyvän kuulon sekä välikorvan säilymisen tulehduksettomana ja ilmapitoisena. Välikorvan rekonstruktiossa on käytetty implanttimateriaaleina perinteisesti potilaan omia kudoksia sekä tarvittaessa erilaisia hajoamattomia biomateriaaleja, mm. titaania ja silikonia. Ongelmana biomateriaalien käytössä voi olla bakteerien adherenssi eli tarttuminen vieraan materiaalin pintaan, mikä saattaa johtaa biofilmin muodostumiseen. Tämä voi aiheuttaa kroonisen, huonosti antibiootteihin reagoivan infektion kudoksessa, mikä usein käytännössä johtaa uusintaleikkaukseen ja implantin poistoon. Maitohappo- ja glykolihappopohjaiset biologisesti hajoavat polymeerit ovat olleet kliinisessä käytössä jo vuosikymmeniä. Niitä on käytetty erityisesti tukimateriaaleina mm. ortopediassa sekä kasvo- ja leukakirurgiassa. Niitä ei ole toistaiseksi käytetty välikorvakirurgiassa. Korvan kuvantamiseen käytetään ensisijaisesti tietokonetomografiaa (TT). TT-tutkimuksen ongelmana on potilaan altistuminen suhteellisen korkealle sädeannokselle, joka kasvaa kumulatiivisesti, jos kuvaus joudutaan toistamaan. Väitöskirjatyö selvittää uuden, aiemmin kliinisessä työssä rutiinisti lähinnä hampaiston ja kasvojen alueen kuvantamiseen käytetyn rajoitetun kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen. Väitöskirjan kahdessa ensimmäisessä osatyössä tutkittiin ja verrattiin kahden kroonisia ja postoperatiivisia korvainfektioita aiheuttavan bakteerin, Staphylococcus aureuksen ja Pseudomonas aeruginosan, in vitro adherenssia titaanin, silikonin ja kahden eri biohajoavan polymeerin (PLGA) pintaan. Lisäksi tutkittiin materiaalien albumiinipinnoituksen vaikutusta adherenssiin. Kolmannessa osatyössä tutkittiin eläinmallissa PLGA:n biokompatibiliteettia eli kudosyhteensopivuutta kokeellisessa välikorvakirurgiassa. Chinchillojen välikorviin istutettiin PLGA-materiaalia, eläimiä seurattiin, ja ne lopetettiin 6 kk:n kuluttua operaatiosta. Biokompatibiliteetin arviointi perustui kliinisiin havaintoihin sekä kudosnäytteisiin. Neljännessä osatyössä tutkittiin kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen vertaamalla sen tarkkuutta perinteisen spiraali-TT:n tarkkuuteen. Molemmilla laitteilla kuvattiin ohimo- eli temporaaliluita korvan alueen kliinisesti ja kirurgisesti tärkeiden rakenteiden kuvantumisen tarkkuuden arvioimiseksi. Viidennessä osatyössä arvioitiin myös operoitujen temporaaliluiden kuvantumista kartiokeila-TT:ssa. Bakteeritutkimuksissa PLGA-materiaalin pintaan tarttui keskimäärin korkeintaan saman verran tai vähemmän bakteereita kuin silikonin tai titaanin. Albumiinipinnoitus vähensi bakteeriadherenssia merkitsevästi kaikilla materiaaleilla. Eläinkokeiden perusteella PLGA todettiin hyvin siedetyksi välikorvassa. Korvakäytävissä tai välikorvissa ei todettu infektioita, tärykalvon perforaatioita tai materiaalin esiin työntymistä. Kudosnäytteissä näkyi lievää tulehdusreaktiota ja fibroosia implantin ympärillä. Temporaaliluutöissä rajoitettu kartiokeila-TT todettiin vähintään yhtä tarkaksi menetelmäksi kuin spiraali-TT välikorvan ja sisäkorvan rakenteiden kuvantamisessa, ja sen aiheuttama kertasäderasitus todettiin spiraali-TT:n vastaavaa huomattavasti vähäisemmäksi. Kartiokeila-TT soveltui hyvin välikorvaimplanttien ja postoperatiivisen korvan kuvantamiseen. Tulokset osoittavat, että PLGA on välikorvakirurgiaan soveltuva, turvallinen ja kudosyhteensopiva biomateriaali. Biomateriaalien pinnoittaminen albumiinilla vähentää merkittävästi bakteeriadherenssia niihin, mikä puoltaa pinnoituksen soveltamista implanttikirurgiassa. Kartiokeila-TT soveltuu korvan alueen kuvantamiseen. Sen tarkkuus kliinisesti tärkeiden rakenteiden osoittamisessa on vähintään yhtä hyvä ja sen potilaalle aiheuttama sädeannos pienempi kuin nykyisen korva-spiraali-TT:n. Tämä tekee menetelmästä spiraali-TT:aa potilasturvallisemman vaihtoehdon erityisesti, jos potilaan tilanne vaatii seurantaa ja useampia kuvauksia, ja jos halutaan kuvata rajoitettuja alueita uni- tai bilateraalisesti.

3D QSAR analysis of oxazolidinone antibacterials: can we predict?

Three-dimensional QSAR studies for substituted aryloxazolidinones 3–9 were conducted using TSAR 3.3. The in vitro activities (MICs) of the compounds against Staphylococcus aureus and Enterococcus faecalis exhibited a good correlation with the prediction made by the model using heat of formation and LUMO energies.

Synthesis, spectral characterization, in-vitro microbiological evaluation and cytotoxic activities of novel macrocyclic bis hydrazone

A macrocyclic hydrazone Schiff base was synthesized by reacting 1,4-dicarbonyl phenyl dihydrazide with 2,6-diformyl-4-methyl phenol and a series of metal complexes with this new Schiff base were synthesized by reaction with Co(II), Ni(II) and Cu(II) metal salts. The Schiff base and its complexes have been characterized by elemental analyses, IR, H-1 NMR, UV-vis, FAB mass, ESR spectra, fluorescence, thermal, magnetic and molar conductance data. The analytical data reveal that the Co(II), Ni(II) and Cu(II) complexes possess 2:1 metal-ligand ratios. All the complexes are non-electrolytes in DMF and DMSO due to their low molar conductance values. Infrared spectral data suggest that the hydrazone Schiff base behaves as a hexadentate ligand with NON NON donor sequence towards the metal ions. The ESR spectral data shows that the metal-ligand bond has considerable covalent character. The electrochemical behavior of the copper(II) complex was investigated by cyclic voltammetry. The Schiff base and its complexes have also been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Shigella dysentery, Micrococcus, Bacillus subtilis, Bacillus cereus and Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Penicillium and Candida albicans) by MIC method. The brine shrimp bioassay was also carried out to study their in-vitro cytotoxic properties. (C) 2009 Elsevier Masson SAS. All rights reserved.

Antimicrobial effects of ethanolic extracts from Cuscuta reflexa Roxb. (Convolvulaceae).

Ethanolic whole plant extracts obtained from Cuscuta reflexa Roxb were screened against Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria to evaluate their antimicrobial activity. Of the four concentrations of plant extract tested (200 µg/mL, 300 µg/mL, 400 µg/mL or 500 µg/mL), 500 µg/mL elicited the greatest zones of bacterial inhibition across three of the bacteria. In contrast, the growth of Salmonella typhi was not halted regardless of extract concentration. At 200 µg/mL, only the growth of E. coli was inhibited. Overall, although the greatest antimicrobial activity was demonstrated to be against E. coli at a concentration of 500 µg/mL (24.6±0.24), upon comparison to the other bacteria, both B. cereus and S. aureus educed similar zones of inhibition upon comparison to their positive antibiotic control.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Optical, Field-Emission, and Antimicrobial Properties of ZnO Nanostructured Films Deposited at Room Temperature by Activated Reactive Evaporation

ZnO nanostructures were deposited on flexible polymer sheet and cotton fabrics at room temperature by activated reactive evaporation. Room-temperature photoluminescence spectrum of ZnO nanostructured film exhibited a week intrinsic UV emission and a strong broad yellow-orange visible emission. TEM and HRTEM studies show that the grown nanostructures are crystalline in nature and their growth direction was indentified to be along [002]. ZnO nanostructures grown on the copper-coated flexible polymer sheets exhibited stable field-emissio characteristics with a threshold voltage of 2.74 V/mu m (250 mu A) and a very large field enhancement factor (beta) of 23,213. Cotton fabric coated with ZnO nanostructures show an excellent antimicrobial activity against Staphylococcus aureus bacteria (Gram positive), and similar to 73% reduction in the bacterial population is achieved compared to uncoated fabrics after 4 h in viability. Using a shadow mask technique, we also selectively deposited the nanostructures at room temperature on polymer substrates.