Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio‐derived materials for delivery of dexamethasone‐sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/–HESI–MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts’ surface morphology by field emission‐scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046–1055, 2016     

Pseudomonas oleovorans as a tool in bioconversions of hydrocarbons: growth,morphology and conversion characteristics in different two-phase systems

The bioconversion of hydrocarbons by Pseudomonas oleovorans has been studied in two-phase systems. In these systems, the hydrocarbon substrate is present in sufficient amounts to form the bulk apolar phase. High cell densities (up to 20 mg dry mass per ml water phase) are reached when the apolar phase consists of n-octane, 1-octene or 1-decene. There is considerable cell damage after incubation for 50–70 h. Loss of cell viability and membrane damage as observed by freeze-fracture electron microscopy correlate with a loss of hydrocarbon oxidation, measured as the conversion of 1-octene to 1,2-epoxyoctane. The final yield of oxidized hydrocarbon in the apolar substrate phase can be increased substantially by replacing the damaged cells with freshly grown cells. Yields up to 150 mg 1,2-epoxyoctane per ml 1-octene and up to 20–25 mg 1,2-epoxyoctane per ml culture were obtained with four cycles of the cell renewal procedure. Several other substrates in addition to octene were tested in the optimized two-phase system. Of these, 1-decene was converted into (R)-1,2-epoxydecane with an optical purity of 60%, while allylbenzene was converted into chiral 1,2-epoxy-3-phenylpropane. Some of the future applications of the conversion products are discussed.     

Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.     

Design rules for nanomedical engineering: from physical virology to the applications of virus-based materials in medicine

Physical virology seeks to define the principles of physics underlying viral infections, traditionally focusing on the fundamental processes governing virus assembly, maturation, and disassembly. A detailed understanding of virus structure and assembly has facilitated the development and analysis of virus-based materials for medical applications. In this Physical Virology review article, we discuss the recent developments in nanomedicine that help us to understand how physical properties affect the in vivo fate and clinical impact of (virus-based) nanoparticles. We summarize and discuss the design rules that need to be considered for the successful development and translation of virus-based nanomaterials from bench to bedside.     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δh) and cell energization. It was demonstrated that the phase thickness Δh decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δh and cell diameter d demonstrated that a decrease in the phase thickness Δh could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Qualitative mathematical discussion of different evolutionary states in water transport systems of plants

We shall present several qualitative mathematical models to describe the early evolution of water transport systems in plants. To perform this in a systematic way we apply methods which have been developed in phenomenological synergetics. These methods rest on the fact that it becomes possible to describe the macroscopic behavior of a complex system by a set of control and order parameters when they are suitably identified. Our presentation is addressed to community with interdisciplinary interests.     

'IntraCell' plugin for assessment of intracellular localization of nano-delivery systems and their targeting to the individual organelles

Efficient intracellular targeting of drugs and drug delivery systems (DDSs) is a major challenge that should be overcome to enhance the therapeutic efficiency of biopharmaceuticals and other intracellularly-acting drugs. Studies that quantitatively assess the mechanisms, barriers, and efficiency of intracellular drug delivery are required to determine the therapeutic potential of intracellular targeting of nano-delivery systems. In this study we report development and application of a novel ‘IntraCell’ plugin for ImageJ that is useful for quantitative assessment of uptake and intracellular localization of the drug/DDS and estimation of targeting efficiency. The developed plugin is based on threshold-based identification of borders of cell and of the individual organelles on confocal images and pixel-by-pixel analysis of fluorescence intensities.We applied the developed ‘IntraCell’ plugin to investigate uptake and intracellular targeting of novel endoplasmic reticulum (ER)-targeted delivery system based on PLGA nanoparticles decorated with ER-targeting or control peptides and encapsulating antigenic peptide and fluorescent marker. Decoration of the nanoparticles with peptidic residues affected their uptake and intracellular trafficking in HeLa cells, indicating that the targeting peptide was identified as ER-targeting signal by the intracellular trafficking mechanisms in HeLa cells and that these mechanisms can handle nano-DDS of the size comparable to some intracellular vesicles (hundreds of nanometers in diameter).We conclude that decoration of nanoparticles with peptidic residues affects their intracellular localization and trafficking and can be potentially used for intracellularly-targeted drug delivery. ‘IntraCell’ plugin is an useful tool for quantitative assessment of efficiency of uptake and intracellular drug targeting. In combination with other experimental approaches, it will be useful for the development of intracellularly-targeted formulations with enhanced and controlled drug pharmacological activities, such as delivery of antigenic peptides for anticancer vaccination and for other applications.     

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography-electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography-mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC(18) narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3-10 microg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of -6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC-ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.     

Boolean modeling techniques for protein co-expression networks in systems medicine

Introduction: Application of systems biology/systems medicine approaches is promising for proteomics/biomedical research, but requires selection of an adequate modeling type.

Areas covered: This article reviews the existing Boolean network modeling approaches, which provide in comparison with alternative modeling techniques several advantages for the processing of proteomics data. Application of methods for inference, reduction and validation of protein co-expression networks that are derived from quantitative high-throughput proteomics measurements is presented. It’s also shown how Boolean models can be used to derive system-theoretic characteristics that describe both the dynamical behavior of such networks as a whole and the properties of different cell states (e.g. healthy or diseased cell states). Furthermore, application of methods derived from control theory is proposed in order to simulate the effects of therapeutic interventions on such networks, which is a promising approach for the computer-assisted discovery of biomarkers and drug targets. Finally, the clinical application of Boolean modeling analyses is discussed.

Expert commentary: Boolean modeling of proteomics data is still in its infancy. Progress in this field strongly depends on provision of a repository with public access to relevant reference models. Also required are community supported standards that facilitate input of both proteomics and patient related data (e.g. age, gender, laboratory results, etc.).     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

Recent progresses in biomedical applications of aptamer-functionalized systems

Aptamers, known as “chemical antibodies” are screened via a combinational technology of systematic evolution of ligands by exponential enrichment (SELEX). Due to their specific targeting ability, high binding affinity, low immunogenicity and easy modification, aptamer-functionalized systems have been extensively applied in various fields and exhibit favorable results. However, there is still a long way for them to be commercialized, and few aptamer-functionalized systems have yet successfully entered clinical and industrial use. Thus, it is necessary to overview the recent research progresses of aptamer-functionalized systems for the researchers to improve or design novel and better aptamer-functionalized systems. In this review, we first introduce the recent progresses of aptamer-functionalized systems’ applications in biosensing, targeted drug delivery, gene therapy and cancer cell imaging, followed by a discussion of the challenges faced with extensive applications of aptamer-functionalized systems and speculation of the future prospects of them.     

Mesoporous Silica Nanoparticles for Cancer Therapy: Energy-Dependent Cellular Uptake and Delivery of Paclitaxel to Cancer Cells

Biocompatible mesoporous silica nanoparticles, containing the fluorescence dye fluorescein isothiocyanate (FITC), provide a promising system to deliver hydrophobic anticancer drugs to cancer cells. In this study, we investigated the mechanism of uptake of fluorescent mesoporous silica nanoparticles (FMSN) by cancer cells. Incubation with FMSN at different temperatures showed that the uptake was higher at 37°C than at 4°C. Metabolic inhibitors impeded uptake of FMSN into cells. The inhibition of FMSN uptake by nocodazole treatment suggests that microtubule functions are required. We also report utilization of mesoporous silica nanoparticles to deliver a hydrophobic anticancer drug paclitaxel to PANC-1 cancer cells and to induce inhibition of proliferation. Mesoporous silica nanoparticles may provide a valuable vehicle to deliver hydrophobic anticancer drugs to human cancer cells.     

Immunological techniques used with fungal plant pathogens: aspects of antigens, antibodies and assays for diagnosis

The present review surveys serological work with phytopathogenic fungi. Knowledge of antigenic determinants of fungi and the possibilities and problems of selecting and using fungal structures and metabolic products for the production of specific antibodies are considered. The paper is intended to be a source of practical information and literature for the active research worker.     

Thiourea: the antioxidant of choice for the purification of proteins from phenol-rich plant tissues

Metabisulfite, diethyldithiocarbamate, and thiourea are potent phenoloxidase inhibitors commonly used during the extraction of plant proteins. Their effects on several amino acid derivatives and peptides are reported. Important and pH-dependent changes are induced by metabisulfite in the uv-absorption spectrum of N-acetyltryptophanamide but not of N-acetyltyrosinamide. Similar spectral changes are also induced in tryptophanyl-containing peptides. Neither diethyldithiocarbamate nor thiourea modified the spectral properties of tryptophan or tyrosine. Cysteinyl groups are rapidly modified by diethyldithiocarbamate, especially in the lower pH range, but not by metabisulfite or thiourea. Diethyldithiocarbamate as well as metabisulfite interfere with cysteinyl groups. It is concluded that thiourea is the most appropriate reagent for use as a phenoloxidase inhibitor.     

Immunostaining of keratin and vimentin in epidermis: comparison of different post-embedding immunogold techniques for electron microscopy

The present study compares different post-embedding staining methods, including conventional and low-temperature embedding techniques, for demonstration of the keratin and vimentin cytoskeleton of epidermal cells, applying commercially available polyclonal and monoclonal antibodies. Immunogold staining (5-nm particles) was performed on the following material: (a) osmium-fixed and Durcupan-embedded material, etched with various solutions; (b) aldehyde-fixed material embedded in Lowicryl K4M at 4 degrees C and -35 degrees C; (c) aldehyde-fixed material embedded in Lowicryl K11M at -60 degrees C with and without cryoprotection with glycerol. In conventionally embedded material we failed to demonstrate intermediate filaments, whereas they were stained after low-temperature embedding with Lowicryl, i.e., K4M 4 degrees C, K4M -35 degrees C, and K11M -60 degrees C. The keratin and vimentin cytoskeleton reacted exclusively with polyclonal antibodies. The best results for antigenicity as well as structural preservation were achieved by post-embedding staining of K4M -35 degrees C-embedded material. Antibodies to keratin stained the cytoskeleton in keratinocytes of all epidermal layers. Filaments were decorated in a linear alignment. Antibodies to vimentin stained the cytoskeleton of Langerhans cells and melanocytes. In these cells a linear distribution pattern of the reaction product along the filaments and an extrafilamentous cluster formation were observed, indicating staining of vimentin and a vimentin-associated protein.     

Comparison of different analysis techniques for the determination of muscle onset in individuals with patellofemoral pain syndrome

To understand patellofemoral pain syndrome (PFPS), recent studies have focused on assessing the onset in the vastus medialis and vastus lateralis to determine whether there is a delay between these muscles’ activation. However, the results of these studies are not in agreement, as some research shows that there is a delay in the VMO, while others do not show delay. It has been suggested that this discrepancies may be due to differences in the signal processing and analysis. For this reason, this study aimed to compare the three techniques used for onset determination – automatic detection, visual inspection and cross-correlation – and to verify whether these methods are able to detect PFPS. The surface electromyography evaluation procedure was conducted in 22 pain-free control individuals and 11 with PFPS diagnoses, during a stair climbing. The standard error of measurement (SEM) showed that cross-correlation presents the lower variation (2.56/3.27, control/PFPS) in relation to visual (3.77/10.19, control/PFPS) and automatic detection (43.23/51.98, control/PFPS, respectively). But when using the cross-correlation technique, we were not able to distinguish the groups (−6.56/−9.74 ms, control/PFPS, p = 0.15). Therefore, use of muscle onset may not be the best way to distinguish individuals with PFPS.