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High-throughput sequencing of eukaryotic, viral, and bacterial genomes provides a huge database of proteins with potential for structure-function analysis. In response to this opportunity, structural genomics projects have been initiated world-wide with the aim of establishing high-throughput structure determination on a genome-wide scale. Crucial to this effort has been the development of protein production technologies for the high-throughput cloning, expression, and purification of proteins. Large-scale structural genomic projects were initiated in the US and Europe, and all have emphasized parallel processing, both in terms of molecular cloning, expression, and purification, driven by the need to accommodate relatively large numbers of potential targets for structural biology at an acceptable cost. This has led to varying degrees of automation and most of the groups involved have set up semiautomated liquid handling systems to carry out some or all of their protocols. However, the protocols can equally well be carried out manually with appropriate equipment, for example multichannel pipette dispensers. The motivation to implement automation is largely to enable processes to be scaleable and sustainable as error-free operations. This chapter reviews the technical developments that have come from structural proteomics and provides protocols for carrying out cloning, expression, and purification procedures in a relatively high-throughput (HTP) and parallel approach.

Interest in processes at the nexus of host–microbe–metal interactions has risen as a result of advancements in the study of metallomics, proteomics, and genomics. These emerging fields have given rise to new developments in powerful analytical methods and technology for studying the identity, distribution, quantity, trafficking, fate, and effects of trace metals in biological systems. Applications of these advanced techniques to the study of metabolic cycles are yielding results and have placed scientists at the threshold of major paradigm shifts in our understanding of the relationships between homeostatic mechanisms of trace metals and pathogenesis of infectious diseases. Fields present at this Forum included chemistry, biology/biochemistry, toxicology, nutrition, immunology, microbiology, epidemiology, environmental and occupational health, as well as environmental and veterinary medicine. Participants were tasked with using their knowledge to discuss how the metabolic cycles of trace metals relate to the pathogenesis of disease during infection. The stimulating dialog that ensued covered a wide range of views, insights, and perspectives on current knowledge and raised important open questions that should be addressed by future research initiatives.

This concluding chapter summarizes what we know and what future directions will help our understanding. It argues that greater phylogenetic breadth is required in future studies of amphibians, and that physiology is at the heart of any complete understanding of amphibian declines occurring worldwide. It concludes with a plea for collaboration and integration of different levels of biological organization in future studies.

The role of integrative approaches to supportive care in urology is as controversial today as at any time in the history of medicine. At present, however, people are witnessing the emergence of a new, unified scientific foundation that brings together the best of modern molecular medicine with the bioinformatics of integrative medicine. This chapter outlines a new bioinformatics model of integrative medicine that complements and is consistent with the molecular dynamics of modern medicine on the genomic, proteomic, physiological, and psychological levels. Much of integrative medicine remains controversial because the typical outcome research purporting to validate the top-down approaches of integrative medicine do not include the entire four-stage bioinformatics cycle of modern molecular medicine. Clinical-experimental research programs that document the efficacy of integrative medicine with the currently emerging standards of validation via genomic and proteomic microarray technology are now needed.

This chapter discusses approaches to understanding cellular ageing (senescence) through molecular biology approaches. Current scientific ideas surrounding the biological and evolutionary basis of senescence are discussed in this chapter, as are recent findings that demonstrate a strong contribution of senescent cells to age-related decline in health. A new approach to generating senescent cells is described, which accelerates cell ageing in the laboratory based on understandings of premature ageing human Werner syndrome, as is a proteomics approach to probing cellular senescence. The premise that ageing is a social construct is refuted from a biological basis, and the importance of approaches to tackling cellular senescence in the human body to improve the quality of later life is strongly advocated.

The biosciences are sometimes ‘activated and fashioned in articulation with neoliberal, entrepreneurial modes of participation’, but also, in simultaneous contrast, assembled as an echo of Merton’s CUDOS norms. This chapter asks how actors establish what counts as good and valuable biomedical science, and how they, in practice, establish what are acceptable relations between science and industry. The chapter shows how the studied actors use two main strategies to uphold a difference between science and industry, and proposes to describe these strategies as two different modes of purification: temporal purification and organizational purification. By introducing modes of purification the chapter highlights the multiplicity of strategies that are utilized to fashion acceptable science–industry relations.

The diverse phenotypes exhibited by marine invertebrate larvae are the result of complex gene-environment interactions. Recently, technological advances in molecular biology have enabled large-scale -omics approaches, which can provide a global overview of the molecular mechanisms that shape the larval genotype-phenotype landscape. -omics approaches are facilitating our understanding of larval development and life history evolution, larval response to environmental stress, the larval microbiome, larval physiology and feeding, and larval behavior. These large-scale molecular approaches are even more effective when combined with large-scale environmental monitoring and phenotypic measurements. Current -omics approaches to studying larvae can be improved by the addition of functional genetic analyses and the reporting of natural variation in gene expression between individuals and populations. Systems-level approaches that combine multiple -omics techniques will allow us to explore in fine detail the interactions of environmental and genotypic influences on larval phenotype.

Flow cytometry is a mature technology: Instruments recognizable as having elements of modern flow cytometers date back at least 30 years. There are many good sources for information about the essential features of flow cytometers, how they operate, and how they have been used. For the purposes of this book, it is necessary to know that flow cytometers have fluidic, optical, electronic, computational, and mechanical features. The main function of the fluidic components is to use hydrodynamic focusing to create a stable particle stream in which particles are aligned in single file within a sheath stream, so that the particles can be analyzed and sorted. The main functions of the optical components are to allow the particles to be illuminated by one or more lasers or other light sources and to allow scattered light as well as multiple fluorescence signals to be resolved and be routed to individual detectors. The electronics coordinate these functions, from the acquisition of the signals (pulse collection, pulse analysis, triggering, time delay, data, gating, detector control) to forming and charging individual droplets, and to making sort decisions. The computational components are directed at postacquisition data display and analysis, analysis of multivariate populations and multiplexing assays, and calibration and analysis of time-dependent cell or reaction phenomena. Mechanical components are now being integrated with flow cytometers to handle plates of samples and to coordinate automation such as the movement of a cloning tray with the collection of the droplets. The reader is directed to a concise description of these processes in Robinson’s article in the Encyclopedia of Biomaterials and Biomedical Engineering. This book was conceived of to provide a perspective on the future of flow cytometry, and particularly its application to biotechnology. It attempts to answer the question I heard repeatedly, especially during my association with the National Institutes of Health–funded National Flow Cytometry Resource at Los Alamos National Laboratory: What is the potential for innovation in flow cytometer design and application? This volume brings together those approaches that identify the unique contributions of flow cytometry to the modern world of biotechnology.

The field of fungal diagnostics encompasses tests that are performed to help diagnose fungal disease, guide its management, and or monitor the effectiveness of its treatment. For some superficial skin and yeast infections, a clinical examination of the patient combined with microscopic examination of the sample may be sufficient to determine that fungal disease is present, even if the specific fungal pathogen is not identified. For deep- seated and systemic infections, a combination of diagnostic tests may be required in order to obtain a definitive diagnosis. These include microscopy to detect fungal elements, culture, detection of circulating antigens and antibodies, and molecular tests. More recently, molecular and proteomic approaches have increasingly dominated the conventional identification of pathogenic yeasts and, to some extent, filamentous fungi, since traditional methods are time consuming. More importantly, conventional methodologies have failed to identify common organisms that display uncharacteristic profiles, or fungal pathogens that are rarely encountered. The ‘gold standard’ for the definitive diagnosis of fungal disease is histology or culture of the fungal pathogen from a clinical specimen. A specimen will routinely be inoculated onto several different types of media, and then incubated at specific conditions and temperatures for up to twenty-one days. Media plates will be examined periodically for growth, and staff will try to identify the fungus using both macroscopic and microscopic morphologies. The few biochemical tests available, e.g. the urease test, can be helpful in identification, most often for yeast species. Microscopy of fungal isolates, histopathological examination of tissue, and fungal specific stains play fundamental roles in the diagnosis of infection for the variety of fungi that cause disease. The most common stain for identifying fungal elements from a cultured isolate is lactophenol fuschin/aniline blue stain. Figure 10.1 depicts the fruiting body (conidiophore) of Aspergillus fumigatus species complex, the most prevalent fungal species responsible for invasive aspergillosis (IA) in severely immunocompromised individuals. Figure 10.2 illustrates the phenotype of a three-day old colony. Serological tests are beneficial when non-culture based diagnosis of fungal disease is required. Complement fixation is predominantly used to diagnose endemic mycoses, e.g. coccidioidomycosis, blastomycosis, and histoplasmosis.

Molecular diagnostics in infection generally relate to the detection and/ or characterization of nucleic acid sequences of infectious agents in clinical samples which are used to provide: ● A laboratory diagnosis. ● A means of monitoring patients at risk of developing disease caused by a particular infection. ● A method to predict through genotypic analysis the susceptibility or resistance to appropriate treatments. ● A measurement of the response to therapy. A few key laboratory techniques underpin the majority of molecular diagnostic tests that are currently used in the field of infection, and include: ● Block-based polymerase chain reaction (PCR). ● Real-time PCR, including quantification. ● Strand displacement amplification. ● Transcription mediated amplification. ● DNA sequencing. These can be commercially sourced, which has the advantage of CE marking, or developed in-house, sometimes referred to as laboratory developed tests (LDTs). Whatever the source, the underlying principles are often the same and rigorous evaluation and validation is required for the adoption of any molecular test in the diagnostic laboratory. The majority of molecular diagnostic tests require the amplification of a specific DNA sequence and its subsequent detection by a variety of means. As such, small sequences of DNA from the infectious agent are amplified from a relatively low copy number in the clinical sample. For example, after thirty to forty cycles of PCR, a single copy of a sequence can theoretically be amplified to over a billion copies. This PCR product, commonly termed amplicon, can provide a template for any further testing with the same PCR test and therefore potentially act as a source for false positive results. Molecular diagnostic laboratories have requirements to keep the different stages of the molecular test separate and minimize the risk of amplicon contamination. Most facilities will have a ‘clean PCR laboratory’ that is used to store the clean reagents such as primers, probes, enzyme mastermixes, and no clinical samples, nucleic extracts, or amplification reactions are ever taken into this environment. Another laboratory is used for the nucleic acid extraction of the clinical samples and this environment is often used to set up the PCR reactions.