Bioinformatics in microbial biotechnology – a mini review

The revolutionary growth in the computation speed and memory storage capability has fueled a new era in the analysis of biological data. Hundreds of microbial genomes and many eukaryotic genomes including a cleaner draft of human genome have been sequenced raising the expectation of better control of microorganisms. The goals are as lofty as the development of rational drugs and antimicrobial agents, development of new enhanced bacterial strains for bioremediation and pollution control, development of better and easy to administer vaccines, the development of protein biomarkers for various bacterial diseases, and better understanding of host-bacteria interaction to prevent bacterial infections. In the last decade the development of many new bioinformatics techniques and integrated databases has facilitated the realization of these goals. Current research in bioinformatics can be classified into: (i) genomics – sequencing and comparative study of genomes to identify gene and genome functionality, (ii) proteomics – identification and characterization of protein related properties and reconstruction of metabolic and regulatory pathways, (iii) cell visualization and simulation to study and model cell behavior, and (iv) application to the development of drugs and anti-microbial agents. In this article, we will focus on the techniques and their limitations in genomics and proteomics. Bioinformatics research can be classified under three major approaches: (1) analysis based upon the available experimental wet-lab data, (2) the use of mathematical modeling to derive new information, and (3) an integrated approach that integrates search techniques with mathematical modeling. The major impact of bioinformatics research has been to automate the genome sequencing, automated development of integrated genomics and proteomics databases, automated genome comparisons to identify the genome function, automated derivation of metabolic pathways, gene expression analysis to derive regulatory pathways, the development of statistical techniques, clustering techniques and data mining techniques to derive protein-protein and protein-DNA interactions, and modeling of 3D structure of proteins and 3D docking between proteins and biochemicals for rational drug design, difference analysis between pathogenic and non-pathogenic strains to identify candidate genes for vaccines and anti-microbial agents, and the whole genome comparison to understand the microbial evolution. The development of bioinformatics techniques has enhanced the pace of biological discovery by automated analysis of large number of microbial genomes. We are on the verge of using all this knowledge to understand cellular mechanisms at the systemic level. The developed bioinformatics techniques have potential to facilitate (i) the discovery of causes of diseases, (ii) vaccine and rational drug design, and (iii) improved cost effective agents for bioremediation by pruning out the dead ends. Despite the fast paced global effort, the current analysis is limited by the lack of available gene-functionality from the wet-lab data, the lack of computer algorithms to explore vast amount of data with unknown functionality, limited availability of protein-protein and protein-DNA interactions, and the lack of knowledge of temporal and transient behavior of genes and pathways.     

Magnetic quantum dots in biotechnology – synthesis and applications

Quantum dots (QDs) have great promise in biological imaging, and as this promise is realized, there has been increasing interest in combining the benefits of QDs with those of other materials to yield composites with multifunctional properties. One of the most common materials combined with QDs is magnetic materials, either as ions (e.g. gadolinium) or as nanoparticles (e.g. superparamagnetic iron oxide nanoparticles, SPIONs). The fluorescent property of the QDs permits visualization, whereas the magnetic property of the composite enables imaging, magnetic separation, and may even have therapeutic benefit. In this review, the synthesis of fluorescent–magnetic nanoparticles, including magnetic QDs is explored; and the applications of these materials in imaging, separations, and theranostics are discussed. As the properties of these materials continue to improve, QDs have the potential to greatly impact biological imaging, diagnostics, and treatment.     

Nitrogen fixation in eukaryotes – New models for symbiosis

Background  

Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Most nitrogen exists in the atmosphere, and utilisation of this source is important as a means of avoiding nitrogen starvation. However, the ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes. These symbioses involve a variety of host organisms, including animals, plants, fungi and protists.     

Round cell sarcomas – Biologically important refinements in subclassification

Round cell sarcomas are a heterogeneous group of tumors that often affect children and young adults and, if untreated, often pursue a very aggressive clinical course. Specific subtypes of round cell sarcoma, like Ewing sarcoma or rhabdomyosarcoma, respond to well-defined therapeutic regimens so that proper classification is crucial for appropriate patient management. A subset of round cell sarcomas, however, lack specific clinical, morphologic, and immunophenotypic features and cannot be unequivocally classified based on such features. Systematic application of cytogenetics and molecular genetic techniques has allowed for the identification of an increasing number of genetically defined subgroups within this category of undifferentiated tumors. Although the clinical relevance of these molecular categories is yet to be proven, the systematic identification of lesions that share reproducible biologic, and often morphologic and immunophenotypic features, has great impact in terms of biologic understanding and coherent classification schemes, and will help to guide the potential development of rational new therapies. In this review we discuss the main categories of undifferentiated round cell sarcoma, in relation to Ewing sarcoma and its molecular variants, with particular emphasis on the genetic and biologic features of recently described entities including desmoplastic small round cell tumor and CIC-DUX4 as well as BCOR-CCNB3-associated round cell sarcomas.This article is part of a Directed Issue entitled: Rare Cancers.     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

A relation between TGF-β and mast cell tryptase in experimental emphysema models

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. The aim of this study was to investigate the effect of cigarette smoke exposure on mast cells and mast cell function in vitro and in vivo in order to get further insight in the role of mast cells in the pathogenesis of emphysema. Cigarette smoke conditioned medium (CSM) induced the expression of mast cell tryptase (MMCP-6) in primary cultured mast cells. This tryptase expression was caused by the CSM-stimulated production of TGF-β in culture and neutralization of TGF-β suppressed the CSM-induced expression of tryptase in mast cells. An increase in mast cell tryptase expression was also found in an experimental model for emphysema. Exposure of mice to cigarette smoke increased the number of mast cells in the airways and the expression of mast cell tryptase. In accordance with the in vitro findings, TGF-β in bronchoalveolar lavage fluid of smoke-exposed animals was significantly increased. Our study indicates that mast cells may be a source of TGF-β production after cigarette smoke exposure and that in turn TGF-β may change the tryptase expression in mast cells.     

Multicenter randomized trial of cell therapy in cardiopathies – MiHeart Study

Background

Cardiovascular diseases are the major cause of death in the world. Current treatments have not been able to reverse this scenario, creating the need for the development of new therapies. Cell therapies have emerged as an alternative for cardiac diseases of distinct causes in experimental animal studies and more recently in clinical trials.

Method/Design

We have designed clinical trials to test for the efficacy of autologous bone marrow derived mononuclear cell therapies in four different cardiopathies: acute and chronic ischemic heart disease, and Chagasic and dilated cardiomyopathy. All trials are multicenter, randomized, double-blind and placebo controlled. In each trial 300 patients will be enrolled and receive optimized therapy for their specific condition. Additionally, half of the patients will receive the autologous bone marrow cells while the other half will receive placebo (saline with 5% autologous serum). For each trial there are specific inclusion and exclusion criteria and the method for cell delivery is intramyocardial for the chronic ischemic heart disease and intracoronary for all others. Primary endpoint for all studies will be the difference in ejection fraction (determined by Simpson's rule) six and twelve months after intervention in relation to the basal ejection fraction. The main hypothesis of this study is that the patients who receive the autologous bone-marrow stem cell implant will have after a 6 month follow-up a mean increase of 5% in absolute left ventricular ejection fraction in comparison with the control group.

Discussion

Many phase I clinical trials using cell therapy for cardiac diseases have already been performed. The few randomized studies have yielded conflicting results, rendering necessary larger well controlled trials to test for efficacy of cell therapies in cardiopathies. The trials registration numbers at the NIH registry are the following: Chagasic cardiomyopathy (NCT00349271), dilated cardiomyopathy (NCT00333827), acute myocardial infarction (NCT00350766) and Chronic Ischemic Heart Disease (NCT00362388).     

Cartilage-specific autoimmunity in animal models and clinical aspects in patients – focus on relapsing polychondritis

Relapsing polychondritis is an autoimmune disease in which an inappropriate immune response destroys cartilage. Cartilage of the ears, larynx and nose rather than spine and joint cartilage is affected by a chronic relapsing and erosive inflammation. Several animal models for relapsing polychondritis have been published in which immunization with various cartilage proteins induces a variety of chondritis symptoms that mimic those seen in patients. In this review we describe the collagens, matrilin-1 and cartilage oligomeric matrix protein as potential autoantigens able to trigger the tissue-specific immune response seen both in patients and in animal models for relapsing polychondritis and related autoimmune diseases.     

Reaction wood – a key cause of variation in cell wall recalcitrance in willow

Background

The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.

Results

A pot trial was designed to test if the ‘RW response’ varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.

Conclusions

Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a ‘RW response’) indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

     

Host cell protein analysis in therapeutic protein bioprocessing – methods and applications

The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system. A range of immunospecific and non-specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non-specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale-up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non-specific methods, such as 2D-DIGE and 2D-HPLC combined with MS.