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Biopolymers for sample collection, protection, and preservation. - Applied microbiology and biotechnology
One of the principal challenges in the collection of biological samples from air, water, and soil matrices is that the target agents are not stable enough to be transferred from the collection point to the laboratory of choice without experiencing significant degradation and loss of viability. At present, there is no method to transport biological samples over considerable distances safely, efficiently, and cost-effectively without the use of ice or refrigeration. Current techniques of protection and preservation of biological materials have serious drawbacks. Many known techniques of preservation cause structural damages, so that biological materials lose their structural integrity and viability. We review applications of a novel bacterial preservation process, which is nontoxic and water soluble and allows for the storage of samples without refrigeration. The method is capable of protecting the biological sample from the effects of environment for extended periods of time and then allows for the easy release of these collected biological materials from the protective medium without structural or DNA damage. Strategies for sample collection, preservation, and shipment of bacterial, viral samples are described. The water-soluble polymer is used to immobilize the biological material by replacing the water molecules within the sample with molecules of the biopolymer. The cured polymer results in a solid protective film that is stable to many organic solvents, but quickly removed by the application of the water-based solution. The process of immobilization does not require the use of any additives, accelerators, or plastifiers and does not involve high temperature or radiation to promote polymerization.
Layer-by-layer assembled carbon nanotube-acetylcholinesterase/biopolymer renewable interfaces: SPR and electrochemical characterization. - Langmuir : the ACS journal of surfaces and colloids
Developing simple, reliable, and cost-effective methods of renewing an inhibited biocatalyst (e.g., enzymatic interfaces) on biosensors is needed to advance multiuse, reusable sensor applications. We report a method for the renewal of layer-by-layer (LbL) self-assembled inhibition-based enzymatic interfaces in multiwalled carbon nanotube (MWCNT) armored acetylcholinesterase (AChE) biosensors. The self-assembly process of MWCNT dispersed enzymes/biopolymers was investigated using surface plasmon resonance (SPR). The LbL fabrication consisted of alternating cushion layers of positively charged CNT-polyethylenimine (CNT-PEI) and negatively charged CNT-deoxyribonucleic acid (CNT-DNA) and a functional interface consisting of alternating layers of CNT-PEI and negatively charged CNT-acetylcholine esterase (CNT-AChE, pH 7.4). The observed SPR response signal increased while assembling the different layers, indicating the buildup of multiple layers on the Au surface. A partial desorption of the top enzymatic layer in the LbL structure was observed with a desorption strategy employing alkaline treatment. This indicates that the strong interaction of CNT-biopolymer conjugates with the Au surface was a result of both electrostatic interactions between biopolymers and the surface binding energy from CNTs: the closer the layers are to the Au surface, the stronger the interactions. In contrast, a similar LbL assembly of soluble enzyme/polyelectrolytes resulted in stronger desorption on the surface after the alkaline treatment; this led to the investigation of AChE layer removal, permanently inhibited after pesticide exposure on glassy carbon (GC) electrodes, while keeping the cushion layers intact. The desorption strategy permitted the SPR and electrochemical electrode surfaces to be regenerated multiple times by the subsequent self-assembly of fresh PEI/AChE layers. Flow-mode electrochemical amperometric analysis demonstrated good stability toward the determination of acetylcholine with 97.1 Â± 2.7% renewability. Our simple, inexpensive approach shows the potential of renewable LbL self-assembled functional interfaces for multiple uses in a wide field of applications such as biosensing, various biotechnological processes, and the food and health industries.
Association of type 2 diabetes mellitus with plasma organochlorine compound concentrations. - Journal of exposure science & environmental epidemiology
The increased prevalence of type 2 diabetes mellitus (T2DM) is associated with obesity, age, and sedentary lifestyle, but exposure to some organochlorine (OC) compounds has also been recently implicated. The hypothesis tested is that higher concentrations of bioaccumulative OC compounds are associated with T2DM. Plasma samples were obtained from a cross-section of adult male and female Caucasians and African Americans, either with or without T2DM from two US Air Force medical facilities. A method of extracting OC compounds from human plasma using solid phase extraction was developed, and three OC compounds [p,p'-DDE (DDE), trans-nonachlor, and oxychlordane] were quantified by gas chromatography/mass spectrometry. Multivariable logistic regression modeling indicated that increasing body mass index (BMI) was associated with T2DM in Caucasians but not in African Americans, and African Americans were more likely to have T2DM than Caucasians with decreasing odds ratios as BMI increased. An association between T2DM and increasing plasma DDE (adjusted for age, base, race, and BMI) was observed. Increasing DDE concentrations were associated with T2DM in older individuals and those with lower BMIs. Thus, in this study sample there was a higher risk of T2DM with increasing DDE concentrations in older people of normal weight and relatively lower risk associated with increasing DDE concentrations in those who are overweight or obese.Journal of Exposure Science and Environmental Epidemiology advance online publication, 22 October 2014; doi:10.1038/jes.2014.69.
Bacteriophage biosensors for antibiotic-resistant bacteria. - Expert review of medical devices
An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.
Biosensor for detection of antibiotic resistant Staphylococcus bacteria. - Journal of visualized experiments : JoVE
A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species (1,2). The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique (3). The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.
Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements. - Journal of microbiological methods
Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.Copyright Â© 2012 Elsevier B.V. All rights reserved.
Key components of the mode of action for hemangiosarcoma induction in pregabalin-treated mice: evidence of increased bicarbonate, dysregulated erythropoiesis, macrophage activation, and increased angiogenic growth factors in mice but not in rats. - Toxicological sciences : an official journal of the Society of Toxicology
In carcinogenicity studies, pregabalin increased hemangiosarcoma incidence in mice but not in rats. Investigative studies, ranging in length from 24 h to 12 months, were conducted in mice (1000 or 5000 mg/kg) and rats (900 mg/kg) to evaluate a potential mode-of-action scheme for tumor formation. Three areas were evaluated: (1) hematopoiesis (because endothelial and hematopoietic cells arise from the same precursor and hemangiosarcomas are primarily located in mouse hematopoietic tissues), (2) angiogenic growth factors (because increased angiogenic growth factors may stimulate vascular tumors), and (3) pulmonary/blood gas parameters (because hypoxia is a known driver for endothelial cell proliferation). In mice, pregabalin rapidly increased platelet and megakaryocyte counts, activated platelets and bone marrow erythrophages, decreased the myeloid-to-erythroid (M:E) ratio (49%), and produced bone marrow and splenic congestion and extramedullary hematopoiesis (EMH). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor immunohistochemical staining were also increased in mouse bone marrow and spleen and vascular endothelial growth factor receptor 2 immunolabeling was increased in liver. Serum bicarbonate was increased within 24 h of pregabalin administration, persisted over time, and was accompanied by decreased respiratory rate (up to 34%) and increased partial pressure of carbon dioxide (pCO(2)), resulting in sustained metabolic alkalosis and elevated blood pH in mice. In contrast, in rats, pregabalin decreased overall bone marrow cellularity, including decreased number of megakaryocytes (24%) with no evidence of erythrophages, no change in M:E ratio, no EMH, and no increase in angiogenic growth factors or blood pH. Persistent alterations in serum bicarbonate, respiratory function, and blood gas parameters in mice, without adequate compensatory mechanisms, has the potential to create chronic tissue hypoxia, an accepted driver of endothelial cell proliferation.
Natural biopolymer for preservation of microorganisms during sampling and storage. - Journal of microbiological methods
Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples. The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus-MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper. Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45Â°C for MRSA and 40-90Â°C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.Copyright Â© 2011 Elsevier B.V. All rights reserved.
Renewable nanocomposite layer-by-layer assembled catalytic interfaces for biosensing applications. - Langmuir : the ACS journal of surfaces and colloids
A novel, easily renewable nanocomposite interface based on layer-by-layer (LbL) assembled cationic/anionic layers of carbon nanotubes customized with biopolymers is reported. A simple approach is proposed to fabricate a nanoscale structure composed of alternating layers of oxidized multiwalled carbon nanotubes upon which is immobilized either the cationic enzyme organophosphorus hydrolase (OPH; MWNT-OPH) or the anionic DNA (MWNT-DNA). The presence of carbon nanotubes with large surface area, high aspect ratio and excellent conductivity provides reliable immobilization of enzyme at the interface and promotes better electron transfer rates. The oxidized MWNTs were characterized by thermogravimetric analysis and Raman spectroscopy. Fourier transform infrared spectroscopy showed the surface functionalization of the MWNTs and successful immobilization of OPH on the MWNTs. Scanning electron microscopy images revealed that MWNTs were shortened during sonication and that LbL of the MWNT/biopolymer conjugates resulted in a continuous surface with a layered structure. The catalytic activity of the biopolymer layers was characterized using absorption spectroscopy and electrochemical analysis. Experimental results show that this approach yields an easily fabricated catalytic multilayer with well-defined structures and properties for biosensing applications whose interface can be reactivated via a simple procedure. In addition, this approach results in a biosensor with excellent sensitivity, a reliable calibration profile, and stable electrochemical response.
Phage Langmuir monolayers and Langmuir-Blodgett films. - Colloids and surfaces. B, Biointerfaces
Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air-water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of âˆ¼40 mN/m at 20 Â°C. The surface pressure-area (Î -A) isotherm possessed a shoulder at âˆ¼7 Ã— 10(4)nm(2)/phage particle, attributed to a change in phage orientation at the air-water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Î -A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air-water interface at low surface pressures with an excluded area per phage particle of 4.6 Ã— 10(4)nm(2). At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5 Ã— 10(3)nm(2)/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air-water interface at a constant surface pressure of 18 mN/m by Langmuir-Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Î“) in Langmuir-Blodgett (LB) films measured by SEM was consistent with that calculated independently from Î -A isotherms at the transfer surface pressure of 18 mN/m (Î“=23 phage particles/Î¼m(2)). The 50 nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air-water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.Copyright Â© 2010 Elsevier B.V. All rights reserved.
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