Molecular microbial ecology: land of the one-eyed king

Studies of microbial biodiversity have made astounding discoveries of late due to the use of methodologies based on phylogenetic analyses of small subunit ribosomal RNA sequences. Although there are limitations to these methods, they can nonetheless be very useful if these limitations are kept in mind. These limitations range from technical problems such as obtaining representative genomic DNA and suitable primers, to conceptual problems such as defining and using meaningful taxonomic units of diversity (species). Here we discuss several of the limitations inherent in studies of microbial diversity that must be considered when interpreting the results obtained using these approaches.     

Prediction of protein retention in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a powerful technique for protein separation. This review examines methodologies for predicting protein retention time in HIC involving elution with salt gradients. The methodologies discussed consider three-dimensional structure data of the protein and its surface hydrophobicity. Despite their limitations, the methods discussed are useful in designing purification processes for proteins and easing the tedious experimental work that is currently required for developing purification protocols.     

Autoradiographical and immunohistochemical analysis of receptor localization in the central nervous system

Summary Quantitative receptor autoradiographic methods have been widely used over the past two decases. Some of the advantages and limitations of these techniques are reviewed here. Comparison with immunohistochemical andin situ hybridization methods is also highlighted, as well as the use of these approaches to study receptor gene over-expression in cell lines. Together, data obtained using these various methodologies can provide unique information on the potential physiological roles of a given receptor protein and/or binding sites in various tissues.     

Lipid peroxidation products in biological tissues

Over the past twenty-years of lipid peroxidation research in this laboratory, considerable effort has gone into development of new methods, with emphasis on measurement of lipid-soluble fluorophores and the volatile hydrocarbons ethane and pentane. Application of these and other methods has been made to biological materials and living animals. Although the various methodologies used in lipid peroxidation research do not necessarily measure the same class of products, and although agreement of results is not always 100%, there is substantial documentation of good correlations between measurements; for example, of trace volatile hydrocarbons with thiobarbituric acid-reacting substances, of pentane production with dietary and/or tissue vitamin E content, and of pentane production with lipid-soluble fluorophores accumulated in spleen as a function of oxidant stress. Individual methodologies do have their inherent limitations. However, measurements of multiple products and their correlations have added significantly to the base of information on biological damage and protection by dietary antioxidants against nutritional and toxicological insults to tissues, cells, and macromolecules as a result of peroxidative and oxidative reactions.     

Methods for the assessment of human body composition: skinfold thickness and bioelectrical impedance measurements

Research to establish indirect methods of determining human body composition began during the 1940s. Renewed interest in the assessment of human body composition has stimulated the need for a balanced understanding of available methodologies of estimating fat-free mass and fat mass. Subsequently a variety of methods has been introduced. However, attempts to describe the theory and practice of individual methods have been limited. The review summarizes the background and to describe the precision or error of skinfold thickness measurement and to highlight the strengths and the limitations of bioelectrical impedance method.     

Empirical limits for template-based protein structure prediction: the CASP5 example

Most protein structure prediction methods use templates to assist in the construction of protein models. In this paper, we analyse the current state of template-based modelling approaches and reach an estimate of the empirical limits of these methods. Our analysis show that current prediction methods are already reaching these empirical accuracy limits in the easier cases, where finding a close homologue to the native target structure is not a problem. However, we find that even in the absence of alignment errors and using optimal templates, template-based methods have intrinsic limitations, suggesting that other methodologies, such as ab initio procedures, must be used if accuracy is ultimately to be improved.     

Diagnostic value of urinary orotic acid levels: applicable separation methods

Urinary orotic acid determination is a useful tool for screening hereditary orotic aciduria and for differentiating the hyperammonemia disorders which cannot be readily diagnosed by amino acid chromatography, thus reducing the need for enzyme determination in tissue biopsies. This review provides an overview of metabolic aberrations that may be related to increased orotic acid levels in urine, and summarises published methods for separation, identification and quantitative determination of orotic acid in urine samples. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the analysis of urinary specimens are described. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.     

Protein structure prediction: challenging targets for CASP10

Functional characterization of proteins being one of the major issues in molecular biology is still unsolved due to several resource and technical limitations of experimental structure determination methods. A suitable methodology for accurate prediction of protein confirmations simply from sequence is therefore emerging as the primary modeling goal of researchers today. Global blind protein structure prediction summit, entitled Critical Assessment of Structure Prediction (CASP), critically assesses the modeling methodologies, to track our algorithmic path development. But our success is still impeded by incompetent modeling methodologies and several key technical lacunas. There is still a great need to focus some key issues for bridging the major though considered trivial gaps, in the upcoming CASP to pave and demarcate our correct way of developing a consistently accurate prediction methodology in the near future. Major problems resulting in divergence of our predicted models from their actual native states are thus highlighted with suggested more stringent and reliable assessment considerations in the CASP test.     

Protein complex prediction: A survey

Protein complexes are one of the most important functional units for deriving biological processes within the cell. Experimental methods have provided valuable data to infer protein complexes. However, these methods have inherent limitations. Considering these limitations, many computational methods have been proposed to predict protein complexes, in the last decade. Almost all of these in-silico methods predict protein complexes from the ever-increasing protein–protein interaction (PPI) data. These computational approaches usually use the PPI data in the format of a huge protein–protein interaction network (PPIN) as input and output various sub-networks of the given PPIN as the predicted protein complexes. Some of these methods have already reached a promising efficiency in protein complex detection. Nonetheless, there are challenges in prediction of other types of protein complexes, specially sparse and small ones. New methods should further incorporate the knowledge of biological properties of proteins to improve the performance. Additionally, there are several challenges that should be considered more effectively in designing the new complex prediction algorithms in the future. This article not only reviews the history of computational protein complex prediction but also provides new insight for improvement of new methodologies. In this article, most important computational methods for protein complex prediction are evaluated and compared. In addition, some of the challenges in the reconstruction of the protein complexes are discussed. Finally, various tools for protein complex prediction and PPIN analysis as well as the current high-throughput databases are reviewed.     

森林生态价值估算方法研究进展

森林生态效益的重要性日益显现,科学家和各国政府都在关注森林生态价值估算,促进了森林生态价值估算方法的日渐成熟。本文总结了近年来国内外森林生态价值的估算方法,将目前常用的估算方法分为市场类估算法、能值估算法、软件模型法3大类,同时分析了前2种常用方法中存在的替代法的合理性有待验证、忽略多重标准的森林管理策略、欠缺考虑周围影响因素、多种生态价值重复计算以及估算中欠缺考虑稀缺性的局限;其次,重点介绍了能较好解决以上问题的GUMBO、CITYgreen和Invest等软件的特点和优势;最后,在总结当前估算方法研究的基础上,提出中国森林生态估算工作未来的研究方向是深化对森林生态结构和功能关系的认识,提高国外估算方法的适用性,加强生态补偿工作对估算方法的反馈。     

Methods for discovery and characterization of cellulolytic enzymes from insects

Abstract Cellulosic ethanol has been identified as a crucial biofuel resource due to its sustainability and abundance of cellulose feedstocks. However, current methods to obtain glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass. Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic material as a source of metabolic glucose. Even though traditional biochemical methods have been used to identify and characterize these enzymes, the advancement of genomic and proteomic research tools are expected to allow new insights into insect digestion of cellulose. This information is highly relevant to the design of improved industrial processes of biofuel production and to identify potential new targets for development of insecticides. This review describes the diverse methodologies used to detect, quantify, purify, clone and express cellulolytic enzymes from insects, as well as their advantages and limitations.     

Progress in sustainability science: lessons learnt from current methodologies for sustainability assessment: Part 1

Purpose

Sustainability Science (SS) is considered an emerging discipline, applicative and solution-oriented whose aim is to handle environmental, social and economic issues in light of cultural, historic and institutional perspectives. The challenges of the discipline are not only related to better identifying the problems affecting sustainability but to the actual transition towards solutions adopting an integrated, comprehensive and participatory approach. This requires the definition of a common scientific paradigm in which integration and interaction amongst sectorial disciplines is of paramount relevance. In this context, life cycle thinking (LCT) and, in particular, life cycle-based methodologies and life cycle sustainability assessment (LCSA) may play a crucial role. The paper illustrates the main challenges posed to sustainability assessment methodologies and related methods in terms of ontology, epistemology and methodology of SS. The aims of the analysis are twofold: (1) to identify the main features of methodologies for sustainability assessment and (2) to present key aspects for the development of robust and comprehensive sustainability assessment.

Methods

The current debate on SS addressing ontological, epistemological and methodological aspects has been reviewed, leading to the proposal of a conceptual framework for SS. In addition, a meta-review of recent studies on sustainability assessment methodologies and methods, focusing those life cycle based, supports the discussion on the main challenges for a comprehensive and robust approach to sustainability assessment. Starting from the results of the meta-review, we identified specific features of sustainable development-oriented methods: firstly, highlighting key issues towards robust methods for SS and, secondly, capitalising on the findings of each review’s paper. For each issue, a recommendation towards a robust sustainability assessment method is given. Existing limitations of sectorial academic inquiries and proposal for better integration and mainstreaming of SS are the key points under discussion.

Discussion

In the reviewed papers, LCT and its basic principles are acknowledged as relevant for sustainability assessment. Nevertheless, LCT is not considered as a reference approach in which other methods could also find a place. This aspect has to be further explored, addressing the lack of multi-disciplinary exchange and putting the mainstreaming of LCT as a priority on the agenda of both life cycle assessment and sustainability assessment experts. Crucial issues for further developing sustainability assessment methodologies and methods have been identified and can be summarised as follows: holistic and system wide approaches, shift from multi- towards trans-disciplinarity; multi-scale (temporal and geographical) perspectives; and better involvement and participation of stakeholders.

Conclusions

Those are also the main challenges posed to LCSA in terms of progress of ontology, epistemology and methodology in line with the progress of SS. The life cycle-based methodologies should be broadened from comparing alternatives and avoiding negative impacts, to also proactively enhancing positive impacts, and towards the achievement of sustainability goals.     

High-performance capillary electrophoresis: Protein charge estimations and peptide mapping by complexation electrophoresis

Two high-performance capillary electrophoretic (HPCE) methods are presented: The first methodology provides a procedure for estimating the isoelectric points of proteins in the absence of chaotropic agents with charge reversal Micro-Coat capillaries. The second method provides an optimized peptide mapping methodology for protein characterization that employs ion-pairing reagents to optimize the HPCE separation. Advantages and limitations of each methodology are discussed in terms of theory and practical experience. Both methodologies are applicable to a variety of proteins and both enhance our ability to characterize proteins on a molecular level.     

Recovering speciation and extinction dynamics based on phylogenies

Phylogenetic trees of only extant species contain information about the underlying speciation and extinction pattern. In this review, I provide an overview over the different methodologies that recover the speciation and extinction dynamics from phylogenetic trees. Broadly, the methods can be divided into two classes: (i) methods using the phylogenetic tree shapes (i.e. trees without branch length information) allowing us to test for speciation rate variation and (ii) methods using the phylogenetic trees with branch length information allowing us to quantify speciation and extinction rates. I end the article with an overview on limitations, open questions and challenges of the reviewed methodology.     

Preclinical cardio-safety assessment of torsadogenic risk and alternative methods to animal experimentation: The inseparable twins

The last decade has been marked by the withdrawal from the market of several medicines whose use in patients has been associated with the development of torsade de pointes (TdP), a potentially life-threatening polymorphic tachycardia. In a few cases, TdP can degenerate into ventricular fibrillation and lead to sudden death, thus constituting a real problem of public health. The recently finalized ICH S7B guideline defines the prolongation of the QT interval on the electrocardiogram as the best biomarker for predicting the torsadogenic risk of a given compound. However, a growing body of evidence suggests that drugs’ torsadogenic potential may not necessarily be proportional to their ability to prolong the QT interval. It is a dynamic combination of multiple predisposing factors and components rather than a single particular event that can trigger this particular tachycardia. Following recommendations of the guideline, pharmaceutical companies have intensively implemented methodologies to assess the possible risk of QT prolongation and TdP in humans. The main problem in cardiac safety pharmacology is how best to combine the capabilities of different methodologies with their strengths and limitations in order to detect the potential of one molecular entity to induce a lethal arrhythmia of very low clinical incidence. This publication will review the current methodologies, focusing on the alternative methods to animal experimentation, including an overview of cardiac modeling.     

Practical radiation dosimetry across a variety of CBCT devices in Radiology

Cone beam technology is becoming more prominent in Radiology. In our hospital we have an extremity CT, an O-arm and a number of C-arms offering 3D capabilities. Each of these modalities use cone beam CT (CBCT) technology to image the area of interest in one single rotation. Traditional CTDI metrics for radiation dosimetry in CT depend on narrow beam geometry. The relevance of the CTDI as a dose indicator for cone beam scanning is contentious due to underestimation of dose lying outside the standard 100 mm chamber length and CTDI phantoms being of insufficient length.In an attempt to better quantify dose from cone beam scanning, alternative methodologies have been developed which attempt to counter the limitations of CTDI methodologies. In this comparison study we utilised the CBCT methodologies outlined in (i) IAEA Report 5, (ii) EFOMP’s protocol on QC in CBCT and (iii) conventional CTDI measurement and tested them on various CBCT systems used in Radiology. These methods were chosen as they use equipment that is typically available to a diagnostic imaging physicist.We determine that the EFOMP protocol and the conventional CTDI method produce the best estimate of the radiation output for quality control purposes. Our conclusion is that the EFOMP protocol is the fastest and easiest method to measure a CBCT metric but it is not always accessible. For the systems in our hospital we will adopt the EFOMP protocol for open systems (C-arms) and perform CTDI_Vol measurements using conventional techniques on enclosed systems (O-arm and extremity CT).     

Respiratory and cuticular water loss in insects with continuous gas exchange: comparison across five ant species

Respiratory water loss (RWL) in insects showing continuous emission of CO(2) is poorly studied because few methodologies can measure it. Comparisons of RWL between insects showing continuous and discontinuous gas exchange cycles (DGC) are therefore difficult. We used two recently developed methodologies (the hyperoxic switch and correlation between water-loss and CO(2) emission rates) to compare cuticular permeabilities and rates of RWL in five species of ants, the Argentine ant (Linepithema humile) and four common native ant competitors. Our results showed that RWL in groups of ants with moderate levels of activity and continuous gas exchange were similar across the two measurement methods, and were similar to published values on insects showing the DGC. Furthermore, ants exposed to anoxia increased their total water loss rates by 50-150%. These results suggest that spiracular control under continuous gas exchange can be as effective as the DGC in reducing RWL. Finally, the mesic-adapted Argentine ant showed significantly higher rates of water loss and cuticular permeability compared to four ant species native to dry environments. Physiological limitations may therefore be responsible for restricting the distribution of this invasive species in seasonally dry environments.     

To label or not to label: applications of quantitative proteomics in neuroscience research

Proteomics has provided researchers with a sophisticated toolbox of labeling-based and label-free quantitative methods. These are now being applied in neuroscience research where they have already contributed to the elucidation of fundamental mechanisms and the discovery of candidate biomarkers. In this review, we evaluate and compare labeling-based and label-free quantitative proteomic techniques for applications in neuroscience research. We discuss the considerations required for the analysis of brain and central nervous system specimens, the experimental design of quantitative proteomic workflows as well as the feasibility, advantages, and disadvantages of the available techniques for neuroscience-oriented questions. Furthermore, we assess the use of labeled standards as internal controls for comparative studies in humans and review applications of labeling-based and label-free mass spectrometry approaches in relevant model organisms and human subjects. Providing a comprehensive guide of feasible and meaningful quantitative proteomic methodologies for neuroscience research is crucial not only for overcoming current limitations but also for gaining useful insights into brain function and translating proteomics from bench to bedside.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.