In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes

Background  

Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050.     

Real time in vitro studies of doxorubicin release from PHEMA nanoparticles

Background  

Many anticancer agents have poor water solubility and therefore the development of novel delivery systems for such molecules has received significant attention. Nanocarriers show great potential in delivering therapeutic agents into the targeted organs or cells and have recently emerged as a promising approach to cancer treatments. The aim of this study was to prepare and use poly-2-hydroxyethyl methacrylate (PHEMA) nanoparticles for the controlled release of the anticancer drug doxorubicin.     

Synthesis of rhamnolipid biosurfactant and mode of hexadecane uptake by Pseudomonas species

Background  

Microorganisms have devised ways by which they increase the bioavailability of many water immiscible substrates whose degradation rates are limited by their low water solubility. Hexadecane is one such water immiscible hydrocarbon substrate which forms an important constituent of oil. One major mechanism employed by hydrocarbon degrading organisms to utilize such substrates is the production of biosurfactants. However, much of the overall mechanism by which such organisms utilize hydrocarbon substrate still remains a mystery.     

OnEX: Exploring changes in life science ontologies

Background  

Numerous ontologies have recently been developed in life sciences to support a consistent annotation of biological objects, such as genes or proteins. These ontologies underlie continuous changes which can impact existing annotations. Therefore, it is valuable for users of ontologies to study the stability of ontologies and to see how many and what kind of ontology changes occurred.     

New resampling method for evaluating stability of clusters

Background  

Hierarchical clustering is a widely applied tool in the analysis of microarray gene expression data. The assessment of cluster stability is a major challenge in clustering procedures. Statistical methods are required to distinguish between real and random clusters. Several methods for assessing cluster stability have been published, including resampling methods such as the bootstrap.     

Pyrene fate affected by humic acid amendment in soil slurry systems

Background  

Humic acid (HA) has been found to affect the solubility, mineralization, and bound residue formation of polycyclic aromatic hydrocarbons (PAHs). However, most of the studies on the interaction between HA and PAH concentrated on one or two of the three phases. Few studies have provided a simple protocol to demonstrate the overall effects of HA on PAH distribution in soil systems for all three phases.     

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

Background  

The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.     

Evidence for the adaptation of protein pH-dependence to subcellular pH

Background  

The availability of genome sequences, and inferred protein coding genes, has led to several proteome-wide studies of isoelectric points. Generally, isoelectric points are distributed following variations on a biomodal theme that originates from the predominant acid and base amino acid sidechain pKas. The relative populations of the peaks in such distributions may correlate with environment, either for a whole organism or for subcellular compartments. There is also a tendency for isoelectric points averaged over a subcellular location to not coincide with the local pH, which could be related to solubility. We now calculate the correlation of other pH-dependent properties, calculated from 3D structure, with subcellular pH.     

Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive <Emphasis Type="Italic">Escherichia coli</Emphasis>protein,RpoS

Background  

The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione-S-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study.     

Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy

Background  

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility.     

Serum IgE reactivity profiling in an asthma affected cohort

Background

Epidemiological evidence indicates that atopic asthma correlates with high serum IgE levels though the contribution of allergen specific IgE to the pathogenesis and the severity of the disease is still unclear.

Methods

We developed a microarray immunoassay containing 103 allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals belonging to families whose members have a documented history of asthma and atopy. We employed k-means clustering, to investigate whether a particular IgE reactivity profile correlated with asthma and other atopic conditions such as rhinitis, conjunctivitis and eczema.

Results

Both case-control and parent-to-siblings analyses demonstrated that while the presence of specific IgE against individual allergens correlated poorly with pathological conditions, particular reactivity profiles were significantly associated with asthma (p<10E-09). An artificial neural network (ANN)-based algorithm, calibrated with the profile reactivity data, correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Multivariate statistical analysis demonstrated that the familiar relationships of the study population did not affect the observed correlations.

Conclusions

These findings indicate that asthma is a higher-order phenomenon related to patterns of IgE reactivity rather than to single antibody reactions. This notion sheds new light on the pathogenesis of the disease and can be readily employed to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile.     

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

Background  

The analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.     

The impact of metabolic syndrome and endothelial dysfunction on exercise‐induced cardiovascular changes

Objective:

There is limited information regarding the synergistic or additive effects of metabolic syndrome (MS) and endothelial dysfunction (ED) on cardiovascular disease (CVD). Altered cardiovascular responses to exercise have been shown to predict future cardiovascular events as well as assess autonomic function. The present study evaluated the impact of MS and brachial artery reactivity (a proxy of ED) on peak exercise‐induced cardiovascular changes.

Design and Methods:

Individuals (n = 303) undergoing a standard nuclear medicine exercise stress test were assessed for MS. Participants underwent a Forearm Hyperaemic Reactivity test and were considered to have dysfunctional reactivity if their rate of uptake ratio (RUR) was <3.55. Resting and peak blood pressure (BP) and heart rate (HR) were measured. Reactivity was calculated as the difference between peak and resting measures.

Results:

Analyses, adjusting for age, sex, resting HR, total metabolic equivalents (METs), and a history of major CVD, revealed a main effect of MS (F = 5.51, η² = 0.02, P = 0.02) and RUR (F = 6.69, η² = 0.02, P = 0.01) on HR reactivity, such that patients with MS and/or poor RUR had reduced HR reactivity. There were no interactive effects of RUR and MS. There were no effects of RUR or MS on systolic BP (SBP) or diastolic BP (DBP) reactivity or rate pressure product (RPP) reactivity.

Conclusions:

The presence of decreased HR reactivity among participants with MS or poor brachial artery reactivity, combined with the lack of difference in other exercise‐induced cardiovascular changes, indicates that these patients may have some degree of parasympathetic dysregulation. Further longitudinal studies are needed to understand the long‐term implications of MS and endothelial abnormalities in this context.     

Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability

Background

We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions.

Methods

Polypeptide ligands of cell surface molecules, including single‐chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity.

Results

A major limiting factor for recovering viable recombinant Ad5 carrying fiber‐fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber‐ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number.

Conclusions

Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber‐ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors. Copyright © 2002 John Wiley & Sons, Ltd.

     

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

Background  

In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol.     

Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

Background  

Shark heavy chain antibody, also called new antigen receptor (NAR), consists of one single Variable domain (V_H), containing only two complementarity-determining regions (CDRs). The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs) make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences.     

The evolutionary history of sharp- and blunt-snouted lenok (<Emphasis Type="Italic">Brachymystax lenok</Emphasis> (Pallas, 1773)) and its implications for the paleo-hydrological history of Siberia

Background  

Broad-scale phylogeographic studies of freshwater organisms provide not only an invaluable framework for understanding the evolutionary history of species, but also a genetic imprint of the paleo-hydrological dynamics stemming from climatic change. Few such studies have been carried out in Siberia, a vast region over which the extent of Pleistocene glaciation is still disputed. Brachymystax lenok is a salmonid fish distributed throughout Siberia, exhibiting two forms hypothesized to have undergone extensive range expansion, genetic exchange, and multiple speciation. A comprehensive phylogeographic investigation should clarify these hypotheses as well as provide insights on Siberia's paleo-hydrological stability.     

Personality may influence reactivity to stress

Background  

Possible mechanisms behind psychophysiological hyperreactivity may be located at a cognitive-emotional level. Several personality traits have been associated with increased cardiovascular reactivity. Subjects with white coat hypertension, which may constitute a kind of hyperreactivity, are found to suppress their emotions and adapt to the surroundings to a larger extent than controls.     

Simultaneous quantitative and allele-specific expression analysis with real competitive PCR

Background  

For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis.     

ErmineJ: Tool for functional analysis of gene expression data sets

Background  

It is common for the results of a microarray study to be analyzed in the context of biologically-motivated groups of genes such as pathways or Gene Ontology categories. The most common method for such analysis uses the hypergeometric distribution (or a related technique) to look for "over-representation" of groups among genes selected as being differentially expressed or otherwise of interest based on a gene-by-gene analysis. However, this method suffers from some limitations, and biologist-friendly tools that implement alternatives have not been reported.