Disruption of urogenital biofilms by lactobacilli

The process that changes a relatively sparse vaginal microbiota of healthy women into a dense biofilm of pathogenic and potentially pathogenic bacteria is poorly understood. Likewise, the reverse step whereby an aberrant biofilm is displaced and returns to a healthy lactobacilli dominated microbiota is unclear. In order to study these phenomena, in vitro experiments were performed to examine the structure of biofilms associated with aerobic vaginosis, urinary tract infections, and bacterial vaginosis (BV). Uropathogenic Escherichia coli were able to form relatively thin biofilms within five days (6 μm height), while Atopobium vaginae and Gardnerella vaginalis formed thicker biofilms 12 μm in height within two days. Challenge of E. coli biofilms with lactobacilli did not result in pathogen displacement. However, the resulting thicker lactobacilli infused biofilms, caused significant E. coli killing. E. coli biofilms challenged with secreted products of L. rhamnosus GR-1 caused a marked decrease in cell density, and increased cell death. Similarly challenge of BV biofilms with lactobacilli infiltrated BV biofilms and caused bacterial cell death. Metronidazole produced holes in the biofilm but did not eradicate the organisms. The findings provide some evidence of how lactobacilli probiotics might interfere with an aberrant vaginal microbiota, and strengthen the position that combining probiotics with antimicrobials could better eradicate pathogenic biofilms.     

Gelatin based microfluidic devices for cell culture

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. Gelatin, crosslinked with the naturally occurring enzyme transglutaminase is molded to produce microchannels suitable for adherent cell culture and analysis. The autofluorescence of the material was shown to be minimal and within the range of typical background, ensuring utility with analyses using fluorescent dyes and labels would not be affected. Also, normal murine mammary epithelial cells were successfully cultured in the microchannels. The morphology of these adherent epithelial cells was shown to be significantly different for cells grown on rigid tissue culture plastic in either macro- or microscale cultures (even in the presence of a surface coating of gelatin) than those grown on the flexible crosslinked gelatin microchannels. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.     

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.     

Antibacterial activity of silver bionanocomposites synthesized by chemical reduction route

ABSTRACT: BACKGROUND: The aim of this study is to investigate the functions of polymers and size of nanoparticles on the antibacterial activity of silver bionanocomposites (Ag BNCs). In this research, silver nanoparticles (Ag NPs) were incorporated into biodegradable polymers that are chitosan, gelatin and both polymers via chemical reduction method in solvent in order to produce Ag BNCs. Silver nitrate and sodium borohydride were employed as a metal precursor and reducing agent respectively. On the other hand, chitosan and gelatin were added as a polymeric matrix and stabilizer. The antibacterial activity of different sizes of silver nanoparticles was investigated against Gram-positive and Gram-negative bacteria by the disk diffusion method using Mueller-Hinton Agar. RESULTS: The properties of Ag BNCs were studied as a function of the polymer weight ratio in relation to the use of chitosan and gelatin. The morphology of the Ag BNCs films and the distribution of the Ag NPs were also characterized. The diameters of the Ag NPs were measured and their size is less than 20 nm. The antibacterial trait of silver/chitosan/gelatin bionanocomposites was investigated. The silver ions released from the Ag BNCs and their antibacterial activities were scrutinized. The antibacterial activities of the Ag BNC films were examined against Gram-negative bacteria (E. coli and P. aeruginosa) and Gram-positive (S. aureus and M. luteus) by diffusion method using Muller-Hinton agar. CONCLUSIONS: The antibacterial activity of Ag NPs with size less than 20 nm was demonstrated and showed positive results against Gram-negative and Gram-positive bacteria. The Ag NPs stabilized well in the polymers matrix.     

Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.     

Application of Oestrogen Receptor Ligand Binding Domain to the Generic Isolation of Oestrogens by Receptor Affinity Chromatography

Human oestrogen receptor (alpha) ligand binding domain (hER-LBD) was expressed in E. coli and isolated using a novel approach. The solubilised recombinant receptor had the expected biological activity in terms of ligand binding affinity and selectivity, indicating the potential for use in the proposed receptor affinity chromatography (RAC) application. Subsequent covalent binding of hER-LBD to agarose support provided an affinity matrix capable of selective binding of oestrogenic ligands, with a capacity for 17β-oestradiol of ～6 ng/mL wet gel. In initial studies, a yield of ～75% of bound ligand from the affinity matrix was obtained by elution with aqueous ethanol. Immobilised hER-LBD eluted with ethanol retained the majority of its capability to bind 17β-oestradiol (E2), indicating the possibility of reuse of the receptor matrix. In ligand-receptor displacement studies, using [³H]E2-saturated immobilised hER-LBD, direct extraction of the xenoestrogen 2′,3′,4′,5′-tetrachloro-4-biphenylol (TeCBol) from a model food (aqueous gelatin solution) was inhibited at the highest concentration of gelatin tested (1%), however, prior precipitation and extraction with ethanol enabled dose dependent binding of TeCBol. The present studies thus provide preliminary proof of principle for the application of hER-LBD for the purpose of RAC and for the generic extraction of oestrogens and xenoestrogens from biological matrices.     

Antibody-based immobilization of bioluminescent bacterial sensor cells

Whole-cell luminescent bioreporter sensors based on immobilized recombinant Escherichia coli are described and evaluated. The sensors were prepared by glutaraldehyde-anchoring of nonspecific anti-E. coli antibodies on aminosylilated gold or silica glass surfaces with subsequent attachment of the probe bacteria. We demonstrate the generality of the concept by attachment of several E. coli strains that express luciferase in response to different physiological stress conditions including heat shock, DNA damage (SOS), fatty acid availability, peroxide and oxidative stress. The sensors can be used either as single- or multiple-use disposable sensing elements or for continuous operation. We show compatibility with optical fiber technology. Storage stability of the sensors exceeded 5 months with no measurable deterioration of the signal. Repeatability on exposure in successive days was <15%, as was sensor to sensor reproducibility. Sensitivity and detection limits of the immobilized cells were comparable to that of non-immobilized bacteria.     

An Extracellular Matrix‐Mimicking Hydrogel for Full Thickness Wound Healing in Diabetic Mice

An extracellular matrix‐mimicking hydrogel is developed consisting of a hyaluronan‐derived component with anti‐inflammatory activity, and a gelatin‐derived component offering adhesion sites for cell anchorage. The in situ‐forming hyaluronan‐gelatin (HA‐GEL) hydrogel displays a sponge‐like microporous morphology. Also, HA‐GEL shows a rapid swelling pattern reaching maximum weight swelling ratio within 10 min, while at the equilibrium state, fully swollen hydrogels display an exceedingly high water content with ≈2000% of the dry gel weight. Under typical 2D cell culture conditions, murine 3T3 fibroblasts adhere to, and proliferate on top of the HA‐GEL substrates, which demonstrate that HA‐GEL provides a favorable microenvironment for cell survival, adhesion, and proliferation. In vivo healing study further demonstrates HA‐GEL as a viable and effective treatment option to improve the healing outcome of full thickness wounds in diabetic mice by effectively depleting the inflammatory chemokine monocyte chemoattractant protein‐1 in the wound bed.     

Adaptation to UVA radiation of E. coli growing in continuous culture

Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50W/m(2). Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7h(-1), 0.5h(-1) and 0.3h(-1) were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells (D=0.05h(-1)) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3h(-1) only a small fraction (1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.     

Quantitative zymography of matrix metalloproteinases by measuring hydroxyproline: application to gelatinases A and B.

Gelatinases A and B are metalloproteinases involved in the degradation of the extracellular matrix. Detection and quantification of these enzymes in physiological and pathological conditions such as rheumatoid arthritis, tumor invasion and metastasis may be clinically useful. Gelatin zymography is an electrophoretic technique specific for gelatinases. It can be used to detect the activity of both the active and latent forms. We have standardized this technique for the active and latent forms of gelatinase A and for the latent form of gelatinase B. We measured the extent of gelatin degradation with an EDC scanning densitometer (Helena). The value recorded was directly proportional to the amount of enzyme. Gelatinase activity was quantified from the gel by assaying hydroxyproline as an index of gelatin breakdown. Gelatin zymography was found to be useful in characterizing gelatinases A and B by their molecular weights and measuring their specific activity by a standardized analysis of the degraded gelatin substrate.     

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.     

Development of Gelatin‐Alginate Hydrogels for Burn Wound Treatment

Hydrogels are interesting as wound dressing for burn wounds to maintain a moist environment. Especially gelatin and alginate based wound dressings show strong potential. Both polymers are modified by introducing photocrosslinkable functionalities and combined to hydrogel films (gel‐MA/alg‐MA). In one protocol gel‐MA films are incubated in alg‐MA solutions and crosslinked afterward into double networks. Another protocol involves blending both and subsequent photocrosslinking. The introduction of alginate into the gelatin matrix results in phase separation with polysaccharide microdomains in a protein matrix. Addition of alg(‐MA) to gel‐MA leads to an increased swelling compared to 100% gelatin and similar to the commercial Aquacel Ag dressing. In vitro tests show better cell adhesion for films which have a lower alginate content and also have superior mechanical properties. The hydrogel dressings exhibit good biocompatibility with adaptable cell attachment properties. An adequate gelatin‐alginate ratio should allow application of the materials as wound dressings for several days without tissue ingrowth.     

Evidence for the mechanism of photocatalytic degradation of the bacterial wall membrane at the TiO2 interface by ATR-FTIR and laser kinetic spectroscopy

The photocatalytic peroxidation of E. coli cell, lipo-polysaccharide (LPS), phosphatidyl-ethanolcholine (PE), and peptidoglycan (PGN) of the E. coli membrane wall has been investigated on TiO2 porous films by ATR-FTIR spectroscopy. The fast reactions of the photogenerated charge carriers in TiO2 with E. coli, LPS, and PE were monitored by laser kinetic spectroscopy. ATR-FTIR spectroscopy allowed the identification of E. coli, LPS, PE, and PGN as photocatalytic peroxidation products. The PGN was observed to be the most resistant membrane wall component. Shorter peroxidation times were observed for LPS and PE. Laser photolysis shows that E. coli, LPS, and PE compete in the scavenging of a surface trapped holes (h+) with the recombination reaction of h+ with the generated electrons (e-) within times > 50 ns. This scavenging leads to the formation of organic radicals initiating the radical chain peroxidation of E. coli, LPS, PE, and PE.     

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

Combined capillary electrophoresis and DNA-fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain-forming cells, where differences in cell size, shape, or chain length may lead to broad, "unfocusable" distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell-specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence-based labeling of whole target cells. In this work, we combined a simple CE-based presence/absence test with FISH to develop a bacterial detection assay having an additional "layer" of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10-15 min), bacterial populations were highly focused ( approximately 2-3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of approximately 300 injected E. coli cells, suggesting the possibility for single-cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE-FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.     

Evaluation of Chitosan-Based Films Containing Gelatin,Chondroitin 4-Sulfate and ZnO for Wound Healing

In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 ± 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.     

Study of the bioeffects of CdTe quantum dots on Escherichia coli cells

Quantum dots (QDs) hold great potential for applications in nanomedicine, however, only a few studies investigate their toxic- and bio-effects. Using Escherichia coli (E. coli) cells as model, we found that CdTe QDs exhibited a dose-dependent inhibitory effect on cell growth by microcalorimetric technique and optical density (OD(600)). The growth rate constants (k) were determined, which showed that they were related to the concentration of QDs. The mechanism of cytotoxicity of QDs was also studied through the attenuated total reflection-fourier transform infrared (ATR-FTIR) spectra, fluorescence (FL) polarization, and scanning electron microscopy (SEM). It was clear that the cell out membrane was changed or damaged by the addition of QDs. Taken together, the results indicated that CdTe QDs have cytotoxic effects on E. coli cells, and this effects might attribute to the damaged structure of the cell out membrane, thus QDs and by-products (free radicals, reactive oxygen species (ROS), and free Cd(2+)) which might enter the cells.     

Biopolymer/Glycopolypeptide‐Blended Scaffolds: Synthesis,Characterization and Cellular Interactions

Three‐dimensional (3D) scaffolds formed from natural biopolymers gelatin and chitosan that are chemically modified by galactose have shown improved hepatocyte adhesion, spheroid geometry and functions of the hepatocytes. Galactose specifically binds to the hepatocytes via the asialoglycoprotein receptor (ASGPR) and an increase in galactose density further improves the hepatocyte proliferation and functions. In this work, we aimed to increase the galactose density within the biopolymeric scaffold by physically blending the biopolymers chitosan and gelatin with an amphiphlic β‐galactose polypeptide (PPO‐GP). PPO‐GP, is a di‐block copolymer with PPO and β‐galactose polypeptide, exhibits lower critical solution temperature and is entrapped within the scaffold through hydrophobic interactions. The uniform distribution of PPO‐GP within the scaffold was confirmed by fluorescence microscopy. SEM and mechanical testing of the hybrid scaffolds indicated pore size, inter connectivity and compression modulus similar to the scaffolds made from 100 % biopolymer. The presence of the PPO‐GP on the surface of the scaffold was tested monitoring the interaction of an analogous mannose containing PPO‐GP scaffold and the mannose binding lectin Con‐A. In vitro cell culture experiments with HepG2 cells were performed on GLN‐GP and CTS‐GP and their cellular response was compared with GLN and CTS scaffolds for a period of seven days. Within three days of culture the Hep G2 cells formed multicellular spheroids on GLN‐GP and CTS‐GP more efficiently than on the GLN and CTS scaffolds. The multicellular spheroids were also found to infiltrate more in GLN‐GP and CTS‐GP scaffolds and able to maintain their round morphology as observed by live/dead and SEM imaging.     

Ion transport and selectivity in model lipid membranes carrying incorporated cytolytic protein toxins

We used artificial planar lipid membranes to investigate, the mode of action of cytolysins of different origin. We studied some pathologically important bacterial toxins (e. g. S. aureus α-toxin, C. perfringens Θ-toxin, B. thuringiensis δ-endotoxin and E. coli α-hemolysin). All these toxins are used by the bacteria to damage the cells of the invaded organism. We also studied cytolysins of animal origin which are used to react against the attack of foreign organisms like cytolysins from the nematocysts of sea anemones. These proteins disrupt the permeability barrier of the attacked cell membrane by opening a pore into the lipid matrix. We found that in most cases a receptor is not truly required to render them competent to bind to a cell membrane, they spontaneously insert into preformed pure lipid membranes. Several properties of the resulting pores were compared. They are generally large, water filled, and stay open for long periods. In most cases neutral molecules up to a few kDa molecular weight (like sugars and metabolites) can easily pass through the channel. They are weakly selective, usually being able to discriminate only between anions and cations. The selectivity depends on the presence of fixed charges on the protein since it is modulated by pH and by chemical modification of the protein charged residues.