from Pierre Amato writing in Cold-Adapted Microorganisms:

Managing biochemical energy is a challenge that microorganisms have to face for maintaining activity at low temperatures, at which metabolic processes are altered by the decrease of reactions rates and by the rigidification of cellular structures, notably enzymes and membranes. Cold-adapted prokaryotes and eukaryotes, while exhibiting distinct trophic modes, share specificities for compensating these thermodynamic effects and keep the cellular machinery running: they elevate the concentration of adenylate compounds, the key molecules of the energy metabolism. This is achieved by tight adjustments acting at several levels of the metabolism in a global strategy of energy saving: strong orientation of the adenylate metabolism toward the production and regeneration of AMP and its phosphorylation, whereas its destruction is repressed; elevation of the respiration rate; intervention of specific enzymes allowing the rapid synthesis of ATP (polyphosphatases and interferases), shifts in the utilization of substrates; and rerouting of central metabolic pathways. These are presented here and illustrated by examples of metabolic regulations in cold-adapted microorganisms evidenced by recent transcriptomic and proteomic approaches.

Further reading: Cold-Adapted Microorganisms

from Takashi Onodera, Katsuaki Sugiura, Shigeru Matsuda and Akikazu Sakudo writing in Prions: Current Progress in Advanced Research:

Although much is known about the effect of PrP^Sc in prion diseases, the normal function of PrP^C is poorly understood. PrP^C may act as an antiapoptotic agent by blocking some of the internal environmental factors that initiate apoptosis. PrP-knockout methods provide powerful hints on the neuroprotective function of PrP^C. Using PrP^C-knockout cell lines, the inhibition of apoptosis through STI1 is mediated by PrP^C-dependent SOD activation. Recently several reports show that PrP^C participate in trans-membrane signaling process associated with hematopoietic stem cell replication and neuronal differentiation. Besides PrP-knockout exhibited wide spread alterations of oscillatory activity in the olfactory bulb as well as altered paired-pulse plasticity at the dendrodendric synapse. Both the behavioral and electro-physiological phenotypes could be rescued by neuronal PrP^C expression. Neuprotein Shadoo (Sho), similarly to PrP^C, can prevent neuronal cell death induced by the expression of PrP△HD mutants, an artificial PrP mutant devoid of internal hydrophobic domain. Sho can efficiently protect cells against exito-toxin-induced cell death by glutamates. Sho and PrP seem to be dependent on similar domains, in particular N-terminal (N) and their internal hydrophobic domain. Sho△N and Sho△HD displayed a reduced stress-protective activity but are complex glycosylated and attached to outer leaflet of the plasma membrane via GPI anchor indicating that impaired activity is not due to incorrect cellular trafficking. In Shadoo over-expressed mice showed large amyloid plaques not seen in wild-type mice. However Shadoo is not a major modulator of PrP^Sc accumulation and scrapie pathogenesis. Sho and PrP share a stress-protective activity. The ability to adopt a toxic conformation of PrP^Sc seems to be specific for PrP.

Further reading: Prions: Current Progress in Advanced Research

from Tamotsu Hoshino, Nan Xiao, Yuka Yajima and Oleg B. Tkachenko writing in Cold-Adapted Microorganisms:

Cold-adapted fungi are widely distributed in the cryopsphere where biosphere is constantly or seasonally covered with snow and/or ice. Fungi normally have different cells in their lifecycle; therefore, thermal dependence of fungal lifecycle stages is completely different from that of bacteria. We showed examples from fungi that the concept of "psychrophile" by Moria in 1975 was not applicable and propose a new term "cryophilic fungi" for those that spend a certain life stage or whole life cycle (sexual and/or asexual reproductions) in cryosphere. Several groups of fungi associated with snow and/or ice, i.e., cryophilc fungi, are illustrated in terms of their ecology, and their ecophysiological adaptation mechanisms to freezing stress are reviewed, here.

Further reading: Cold-Adapted Microorganisms

from Takashi Onodera and Katsuaki Sugiura writing in Prions: Current Progress in Advanced Research:

Results in Netherlands show that classic scrapie control can be obtained at the national scale without a loss of genetic polymorphism from any of sheep breed. No classical scrapie strain thus far has escaped ARR-associated resistance. Ongoing studies show that atypical scrapie strain also was controlled by ARR-associated resistance. In line with this expectation, the breeding program proved successful in Dutch flock in 2010. When considering the rapid outbreak control as observed in Netherland study, the use of resistant rams seems sufficient and can be recommended as a control strategy in scrapie-affected countries. The origin of atypical BSE cases is currently unknown. As with classical BSE, exposure of these animals to feed contaminated with low titers of TSE agent cannot be excluded, although other origins for these TSE forms cannot be discarded. In particular, the unusually old ages of all H-BSE and L-BSE identified cases and their apparent low-prevalence in the population could suggest that these atypical BSE forms are arising spontaneously. PMCA needs to be highly standardized and robust in terms of a consistent and objectively quantifiable PrPres amplification if to be used for quantification of the proteinaceous seeding activity of prions. There is a direct quantitative correspondence between the seeding and infectious activities of 263K scrapie prions measured by RT-QuIC and bioassay. The methodological, conceptual and practical results described in the report of 263K scrapie prions should be validated for the most human TSE agents. Although scrapie has been known for decades, relatively little attention has been paid to it as a natural disease of sheep and goats mainly because the economical impact has been relatively small compared to other diseases in sheep. The occurrence of bovine spongiform encephalopathy (BSE) provide a new impetus to research into the transmissible spongiform encephalopathies (TSE). Not only was the economical impact of BSE much greater than that of scrapie, the link with variant Creutzfeldt-Jakob disease (vCJD) in humans also gave rise to serious concerns regarding food safety.

Further reading: Prions: Current Progress in Advanced Research

from Seiji Yamauchi, Shinsuke Fukuda and Hidenori Hayashi writing in Cold-Adapted Microorganisms:

Psychrophilic microorganisms can optimally grow at temperatures below 15°C. In these microorganisms, heat stress occurs at relatively low temperatures in comparison with that in mesophilic microorganisms. The majority of psychrophilic microorganisms possess genes encoding a complete set of heat shock proteins (Hsps). Therefore, psychrophilic microorganisms respond to heat stress by producing Hsps like other microorganisms; however, they need a specific system to enable the expression and function of Hsps at relatively low temperatures. In this chapter, we summarize the heat shock response of psychrophilic microorganisms, focusing on how this response starts working at relatively low temperatures and what the features of psychrophilic Hsps are.

Further reading: Cold-Adapted Microorganisms

from Martin A. Lee, David J. Squirrell, Dario L. Leslie and Tom Brown writing in Real-Time PCR: Advanced Technologies and Applications:

The development of fluorescent methods for the closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Energy Transfer. In this chapter we describe these methods in detail, outline the principles of each process, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to show how they may be used on various commercially available thermal cyclers.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Keiji Uchiyama and Suehiro Sakaguchi writing in Prions: Current Progress in Advanced Research:

Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in animals, are a group of incurable neurodegenerative disorders caused by proteinaceous infectious agents, the so-called prions. No preventative vaccines and therapeutics of prion diseases have been developed. Recent lines of evidence suggest that antibodies against prion protein might be beneficial for both preventing and treating prion disease. In this chapter, we first discuss the possibility that there might be many individuals who are latently infected with vCJD prions in human populations, and then introduce the so far reported immunological approaches for development of prion vaccines and immunotherapy against prion disease, including our recent work.

Further reading: Prions: Current Progress in Advanced Research

from Hideharu Shintani and Gerald McDonnell writing in Prions: Current Progress in Advanced Research:

Several inactivation procedures to prion and endotoxins are reported so far. Most of these methods are not applicable to re-usable medical devices due to failure of achievement of material and functional compatibility. Gas plasma inactivation procedure for prion and endotoxin was studied and attain both sterility assurance level (SAL) of 10^-6 and material and functional compatibility in ease.

Further reading: Prions: Current Progress in Advanced Research

from Martin A. Lee, David J. Squirrell and Dario L. Leslie writing in Real-Time PCR: Advanced Technologies and Applications:

A range of factors can cause false negative results in real-time PCR through effects on one or more of the reaction components. Consequently applications requiring a high level of confidence need to be designed to control for the occurrence of false negatives. Whilst an external, or batch, control is often used, the ideal control is an internal one included in the reaction cocktail in a multiplex assay. Here we discuss the application and development of molecular mimics as controls in real-time PCR and explain concepts and experimental considerations to aid in the optimisation of controlled multiplexed assays.

Further reading: Real-Time PCR: Advanced Technologies and Applications

Real-Time PCR: Advanced Technologies and Applications Edited by: Nick A. Saunders and Martin A. Lee ISBN: 978-1-908230-22-5 Publisher: Caister Academic Press Publication Date: July 2013 Cover: hardback

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from Ahmad Iskandar Bin Haji Mohd Taha, Rifat Zubair Ahmed, Taro Motoigi, Kentaro Watanabe, Norio Kurosawa and Hidetoshi Okuyama writing in Cold-Adapted Microorganisms:

Ever since Escherichia coli, which is a mesophilic bacterium, was found to adjust its membrane fluidity in a liquid crystalline state by modulating fatty acid composition and the designation of this process as homeoviscous or homeophasic adaptation, numerous analogous phenomena have been reported in cold-adapted bacteria [psychrophilic or psychrotrophic (psychrotolerant) bacteria]. Unsaturation, which includes the biosynthesis of monounsaturated or long-chain polyunsaturated fatty acids and branched fatty acids, and branched-chain formation are the most important types of fatty acid modulation in psychrophilic and psychrotrophic (psychrotolerant) bacteria. The distribution of these fatty acids is not restricted to cold-adapted microorganisms: rather, it appears to depend on bacterial diversity (Gram- positive or negative) and/or habitat (terrestrial or marine environment) than on temperature. Eicosapentaenoic acid, which has been detected only in marine Gram-negative bacteria, had been regarded to confer significant membrane fluidity in bacteria, but it is now considered that it may also have a function in antioxidation or membrane modulation by constraining membrane fluidity. The mode of fatty acid modulation is unlikely to differ between psychrophilic and psychrotrophic bacteria, which had narrower and wider growth temperature ranges, respectively. The sole difference seems to be higher capacity to modulate fatty acid composition in psychrotrophic bacteria than in psychrophilic bacteria.

Further reading: Cold-Adapted Microorganisms

from Jacob Moran-Gilad and Nick Saunders writing in Real-Time PCR: Advanced Technologies and Applications:

There are significant risks associated with the introduction of new diagnostic assays based on real-time PCR. A consistent approach to the management of the development, validation, verification and implementation of such assays is essential to meet good practice. Adoption of a strategic framework which is followed rigorously by the team is important to ensure that the project is successful. The project core team must have clearly defined roles and produce adequate documentation that can be assessed by a separate review team. Project planning should include aspects such as setting clear objectives, identification of materials and the resources required to complete the project. Of particular importance for real-time PCR diagnostics is the inclusion of adequate positive controls. Tools for the selection of appropriate positive controls are presented and discussed here as a key aspect of diagnostic PCR project management. Following the introduction of assays to practice it is vital to maintain the standard operating procedure to ensure that it is followed consistently and so that any necessary changes are documented and adequately validated. The users of diagnostic assays must be aware of the contents of the project dossiers or have other means to verify their provenance and performance. Project documentation should be maintained to ensure that the quality of the validation data is strengthened over time.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Katayama Taiki and Michiko Tanaka writing in Cold-Adapted Microorganisms:

Bacteria and fungi preserved for long periods at sub-zero temperatures in Alaskan and Siberian permafrost ice wedge were reactivated on agar by aerobic cultivation at 15°C. Culturable bacteria differed among ice samples, but several phylogenetic groups were closely related to those in other frozen environments. Incubation under controlled temperatures and the Arrhenius profiles of bacterial isolates from the Alaskan ice wedge indicated that they could grow at temperatures below 0°C without remarkable alterations in their cellular process. The novel ice wedge isolates, Glaciibacter superstes AHU1791T and Tomitella biformata AHU1821T, increased membrane fluidity at lower to subzero temperatures by modulating the fatty acid composition of the cytoplasmic membrane. Reactivating the non-culturable state of ice wedge isolates using resuscitation promoting factor (Rpf) and culturing melted ice wedge with Rpf provided indirect evidence that non-culturable bacteria exist within the ice wedge in situ. Bacteria in ice wedges can change their membrane fatty acid composition and/or structure to survive, but may then lose the ability to grow under laboratory conditions, as a final adaptation to long periods in a frozen natural environment.

Further reading: Cold-Adapted Microorganisms

from Jim Huggett, Tania Nolan and Stephen A. Bustin writing in Real-Time PCR: Advanced Technologies and Applications:

The capacity to amplify and detect trace amounts of nucleic acids has made the polymerase chain reaction (PCR) the most formidable molecular technology in use today. Its versatility and scope was further broadened first with the development of reverse transcription (RT)-PCR, which opened up the entire RNA field to thorough exploration and then, most conspicuously, with its evolution into real-time quantitative PCR (qPCR). Speed, simplicity, specificity, wide linear dynamic range, multiplexing and high throughput potential, reduced contamination risk, simplified detection and data analysis procedures as well as availability of increasingly affordable instrumentation and reduced reagent cost have made qPCR the molecular method of choice when quantifying nucleic acids. Detection of pathogens, SNP analyses and quantification of RNA, even real-time analysis of gene expression in vivo have become routine applications and constant enhancements of chemistries, enzymes, mastermixes and instruments continue to extend the scope of qPCR technology by promising added benefits such as extremely short assay times measured in minutes, low reagent usage and exceptionally rapid heating/cooling rates. The whole process is driven by the insatiable demand for ever-more specific, sensitive, convenient and cost-effective protocols. However, it has also become clear that variable pre-assay conditions, poor assay design and incorrect data analysis have resulted in the regular publication of data that are often inconsistent, inaccurate and often simply wrong. The problem is exacerbated by a lack of transparency of reporting, with the details of technical information wholly inadequate for the purpose of assessing the validity of reported qPCR data. This has serious consequences for basic research, reducing the potential for translating findings into valuable applications and potentially devastating implications for clinical practice. In response, guidelines proposing a minimum standard for the provision of information for qPCR experiments ("MIQE") have been launched. These aim to establish a standard for accurate and reliable qPCR experimental design as well as recommendations to ensure comprehensive reporting of technical detail, indispensable conditions for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Martin Urban and Kim E. Hammond-Kosack writing in Fusarium: Genomics, Molecular and Cellular Biology:

The most destructive phase of the wheat-Fusarium interaction commences at anthesis and results in lower grain yields, reduced grain quality and the contamination of grain with harmful mycotoxins. Current control strategies are often inadequate. Globally, F. graminearum is the most problematic species. A recent microscopic study has revealed a hitherto unsuspected latent phase where hyphae symptomlessly advance the infection through living wheat floral tissues prior to host cell death. Various forward and reverse genetic methods have been developed to explore the repertoire of Fusarium genes contributing to disease formation, mycotoxin production and sporulation. At the time of writing this chapter, 159 genes are known to contribute to virulence. A newly devised seven-stage floral disease assessment key is described to assist in the inter-comparison of mutant phenotypes. Various innovative bioinformatics approaches are currently being used to predict additional virulence components, by taking advantage of the wealth of genomic, transcriptomic, metabolomic and phenotypic knowledge available. These include (1) InParanoid analyses to infer gene function by using the phenomics data sets available for ~100 pathogenic species in the Pathogen-Host Interaction database, (2) the prediction of protein-protein interaction networks, and (3) statistical analysis of the spatial distribution of specific gene types within the genomic landscape and via comparative phytopathogen genome analyses. Soon data arising from various next generation sequencing approaches will increase the precision of both experimental and predictive studies.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

from Hermann C. Altmeppen, Berta Puig, Susanne Krasemann, Clemens Falker, Frank Dohler and Markus Glatzel writing in Prions: Current Progress in Advanced Research:

Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative conditions occurring in humans and animals that may be transmitted. Experimental data and neuropathological examinations show that prions (here defined as the agent responsible for transmissible spongiform encephalopathies) consist of a self-propagating isoform of the cellular prion protein. Nucleic acids are not required for propagation of prions. In the last years a number of questions regarding the mechanism of prion propagation and neurotoxicity as well as the spread of prions to and within the brain have been answered, yet essential pieces of information regarding the execution of cell death and cell-to-cell spread of prions remain to be elucidated.

Further reading: Prions: Current Progress in Advanced Research

from Scott D. Rose, Richard Owczarzy, Joseph R. Dobosy and Mark A. Behlke writing in Real-Time PCR: Advanced Technologies and Applications:

Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodified DNA bases, selective use of chemically-modified bases and non-base modifying groups can prevent primer-dimer artifacts, improve specificity, and allow for selective amplification of sequences that differ by as little as a single base. A wide variety of chemical modifications have been characterized for use in qPCR. As a general class, the modifications that are in greatest use today increase the binding affinity of the oligonucleotides (i.e., increase the melting temperature, Tm). Tm-enhancing modifications allows both primers and probes to be shorter, improving the differential Tm (DTm=Tm match-Tm mismatch) between perfect match and mismatch hybridization. These modifications have widespread application in allele-specific PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base modifications are in common use that decrease specificity and improve duplex formation in the presence of base mismatches. Although these modifications lower Tm, they have less of an impact on primer stability than do actual mismatched bases. Universal bases permit use of primers and probes in polymorphic loci when it is desirable to detect all sequence variants and minimize mismatch discrimination.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Takumi Nishiuchi writing in Fusarium: Genomics, Molecular and Cellular Biology:

Plant pathogenic species of Fusarium produce numerous secondary metabolites during infection of host plants. These metabolites often perturb host defense responses and suppress plant growth. Plant responses to Fusarium metabolites can be classified as follows: (1) inhibition of root or shoot growth; (2) inhibition of seed germination; (3) changes in leaf color such as chlorosis; (4) cell death; and (5) suppression or activation of defense responses. These phytotoxic effects of Fusarium metabolites have been reported in various plant species. Two major Fusarium metabolites, fumonisins and trichothecenes, induce apoptosis-like programmed cell death and can contribute to virulence of fusaria on some plants. Recently, signaling events have been implicated in plant responses to Fusarium metabolites. In contrast, production of the growth-promoting metabolites gibberellins by the rice pathogen Fusarium fujikuroi results in the seedling elongation symptom characteristic of bakanae disease of rice. Thus, Fusarium secondary metabolites have various effects in host plants. This chapter reviews Fusarium secondary metabolites and how plant respond to them.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

from Takashi Onodera and Akikazu Sakud writing in Prions: Current Progress in Advanced Research:

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurological diseases that include Creutzfeldt-Jakob Disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids. A key event in prion diseases is the conversion of the cellular, host-encoded prion protein (PrP^C) to its abnormal isoform (PrP^Sc) predominantly in the central nervous system of the infected host (Aguzzi et al. 2004).The diseases are transmissible under some circumstances, but unlike other transmissible disorders, prion diseases can also be caused by mutations in the host gene. The mechanism of prion spread among sheep and goats that develop natural scrapie is unknown. CWD, transmissible mink encephalopathy (TME), BSE, feline spongiform encephalopathy (FSE), and exotic ungulate encephalopathy (EUE) are all thought to occur after the consumption of prion-infected material. Most cases of human prion disease occur from unknown reasons, and >20 mutation in the prion gene may lead to inherited prion disease. In other instances, prion diseases are contracted by exposure to prion infectivity. This raises the question of how a mere protein aggregate can trespass mucosal barriers, circumvent innate and adoptive immunity, and travel across the blood-brain barrier to eventually provoke brain disease. To start the chapters of this book we will introduce a few topics in current prion studies.

Further reading: Prions: Current Progress in Advanced Research

from Akikazu Sakudo writing in Prions: Current Progress in Advanced Research:

Prion diseases are devastating neurodegenerative disorders caused by infectious proteinaceous agents known as prions. Prion protein (PrP) gene (Prnp)-deficient mice do not infect with prion agent, indicating essential role of PrP for prion diseases. An abnormal isoform of prion protein (PrP), known as PrP^Sc, which is converted from cellular PrP (PrP^C), is thought to constitute the prion agent. Recently, proteins homologous to PrP have been found, suggesting the existence of other PrP family members, which so far include PrP, Doppel (Dpl) and Shadoo (Sho). In this chapter, the author introduces recent research on the physiological function of PrP and PrP-related proteins together with our own studies.

Further reading: Prions: Current Progress in Advanced Research

from Kazuaki Yoshimune, Jun Kawamoto and Tatsuo Kurihara writing in Cold-Adapted Microorganisms:

This chapter primarily describes cold shock proteins (CSPs), which are induced in response to temperature downshift in both psychrophiles and mesophiles. These proteins are important for various cellular processes, including transcription, translation, protein synthesis and folding, and membrane functions, to maintain their viability under cold conditions. Here, the CSPs in psychrophilic and mesophilic microorganisms are represented on the basis of the results of proteome analyses. In particular, CSPs that were isolated from the Antarctic psychrophile Shewanella livingstonensis Ac10 are described in detail. A number of microorganisms induce molecular chaperones in response to the cold, although the majority of chaperones are induced as a result of heat shock. Chaperones with peptidyl prolyl cis-trans isomerase activity are often induced by cold to accelerate protein folding by interconverting the cis and trans isomers of proline imidic peptide bonds, and these cold shock chaperones are also discribed here. Cold adapted proteins often have higher flexibility as compared to mesophilic proteins. The features of cold adapted proteins are briefly described. Finally, the production of recombinant proteins in psychrophiles is shown to suggest a future application of psychrophiles.

Further reading: Cold-Adapted Microorganisms

from Yuichi Nogi writing in Cold-Adapted Microorganisms:

Psychrophilic microorganisms are extremophiles that are capable of growth and reproduction at low temperatures. They are present in marine environments, which occupy slightly more than 70% of Earth's surface, especially in the Arctic, Antarctica, and deep seas at temperatures lower than 15°C. Marine psychrophiles utilize a wide variety of metabolic pathways, including photosynthesis, chemoautotrophy, and heterotrophy. The deep-sea bacteria called psychropiezophiles "love" both high pressure and low temperature. Marine psychrophiles are characterized by lipid cell membranes chemically resistant to hardening in response to the cold. Most psychrophiles are Bacteria, and psychrophily is present in widely diverse microbial lineages within the broad groups of Alpha-, Beta-, Delta-, and Gammaproteobacteria and the Bacteroidetes phylum.

Further reading: Cold-Adapted Microorganisms

from Nick A. Saunders writing in Real-Time PCR: Advanced Technologies and Applications:

Real-time PCR arrays are tools that allow convenient testing of samples in many assays concurrently, parallel testing of many samples or testing of multiple samples and targets simultaneously. It is desirable to standardise and automate primer and probe selection due to the large number of assays that must be designed. Furthermore, it is useful to use probe selection techniques that increase the robustness of the individual assays since this will increase the level of compatibility between the assays and decrease the complexity of interpretation of the outputs. A simple approach to creating real-time PCR arrays is to use microtitre plates which currently have capacities of 96, 384 or 1536 features. Such arrays can be populated with user designed assays or with tests selected form a menu of over one million that are commercially available. A primary application of such arrays has been to verify gene expression data obtained using hybridisation. Cramming additional features into a device of manageable scale has led to the introduction of nanolitre volume arrays that diverge from the microtitre plate pattern. Several thousand different reactions can now be included in a single real-time PCR array. The reduction in scale also has advantages in terms of the volumes of materials required. As real-time arrays are miniaturised the number of pipetting steps required increases and it is often necessary to pre-configure them commercially leading to relative inflexibility. This limitation has prompted the development of arrays that include microfluidic channels and valves. These 'chips' can be loaded via relatively few liquid handling steps to create custom applications.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Kyle R. Pomraning, Lanelle R. Connolly, Joseph P. Whalen, Kristina M. Smith and Michael Freitag writing in Fusarium: Genomics, Molecular and Cellular Biology:

Multiple mechanisms control genome stability in filamentous fungi. To limit the expansion of repeated DNA, e.g. transposable elements (TEs), a group of filamentous ascomycetes make use of a duplication-dependent mutator system, called "Repeat-Induced Point mutation" (RIP). This phenomenon was the first eukaryotic genome defense system identified and characterized in the 1980s by Selker and colleagues in pioneering studies with Neurospora crassa. RIP detects gene-sized duplications and aligns homologous copies by an unknown homology search mechanism during pre-meiosis. Transition mutations (C:G to T:A) are introduced, typically into both or all copies of the DNA segments. In 2007, RIP was first described in Gibberella zeae (anamorph: Fusarium graminearum) by Kistler and colleagues. Here we review previous experiments and add our recent data, which confirm that RIP occurs at relatively high frequencies in this homothallic species. We show that the G. zeae rid homologue is required for RIP, as had been found in N. crassa. In contrast to N. crassa, DNA methylation does not seem to be a common consequence of RIP. Lastly, we discuss potential evolutionary consequences of RIP in heterothallic and homothallic fungi.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

from Francis Trail writing in Fusarium: Genomics, Molecular and Cellular Biology:

Fusarium spp. represent an array of sexual life styles: asexual, homothallic, and heterothallic. The recent availability of genomic resources for several Fusarium species has inspired intense research on these organisms, including a better understanding of sporulation. Although studies have clarified the arrangement of the MAT idiomorphs among these species, little is known about the role of MAT genes in sex and fruiting body development. For most Fusarium species, the sexual cycle does not predominate in the field. However, F. graminearum, a homothallic species, relies on sexual development for spore dissemination to host plants. Recent functional studies have revealed genes involved in many aspects of perithecium development in this species. This chapter will focus on morphogenic aspects of sexual development, summarize the function of genes that have been shown to affect development, and speculate about the ecological and evolutionary implications of sexual life styles.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

from H. Corby Kistler, Martijn Rep and Li-Jun Ma writing in Fusarium: Genomics, Molecular and Cellular Biology:

Fungi in the genus Fusarium have a great negative impact on the world economy, yet also hold great potential for answering many fundamental biological questions. The advance of sequencing technologies has made possible the connection between phenotypes and genetic mechanisms underlying the acquisition and diversification of such traits with economic and biological significance. This chapter provides a historical view of our understanding of genomic structural variation among Fusarium species. Prior to the genomic era, chromosomal variation was observed between Fusarium species and among isolates of F. oxysporum and F. solani (teleomorph Nectria haematococca). Such observations led to the discovery of supernumerary chromosomes in Nectria haematococca MPVI and have established their role in fungal-plant interactions. Contemporary comparative genomic studies not only have confirmed the existence of supernumerary chromosomes in the F. oxysporum and F. solani genomes, but also have provided strong evidence for the horizontal transmission of these chromosomes and their role as genetic determinants of host specific virulence. Overall, knowledge of the highly dynamic Fusarium genomes establishes them as eukaryotic models allowing greater understanding of genome plasticity and adaptive evolution to ecological niches.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

from Chaminda Salgado and Waqar Hussain writing in Real-Time PCR: Advanced Technologies and Applications:

Myriad methods for the extraction and purification of nucleic acids prior to PCR are currently used throughout the community. While these methods have many unique and bespoke aspects, they broadly follow a sequence of lysis, isolation, washing and elution to get from a complex biological sample to purified nucleic acid that can be used in a PCR reaction. Various common methods available for each stage are described and potential sequences for particular sample types can be discerned. The potential for these methods to be automated are discussed and the process options summarized with respect to the speed of the methods, technical skill required and the resultant purity and yield that can be expected.

Further reading: Real-Time PCR: Advanced Technologies and Applications

"this book addresses important themes in the molecular epidemiology of bacterial pathogens ... Another consistent strength of this book is the detailed coverage of antibiotic resistance across the entire spectrum of food and waterborne pathogens ... for clinicians, food or environmental scientists, or public health officials interested in gaining a foundation in the molecular microbiology of this important group of pathogens, this book would provide an excellent foundation. Alternatively, for those with expertise in specific organisms, this book provides an interesting look across the spectrum of food and waterborne bacterial pathogens." from Jason B. Harris (Harvard Medical School, Boston, USA) writing in Clin. Inf. Dis. (2013) read more ...

Foodborne and Waterborne Bacterial Pathogens: Epidemiology, Evolution and Molecular Biology Edited by: Shah M. Faruque ISBN: 978-1-908230-06-5 Publisher: Caister Academic Press Publication Date: July 2012 Cover: hardback "an excellent foundation" (Clin. Inf. Dis.)

from Philipp Wiemann and Bettina Tudzynski writing in Fusarium: Genomics, Molecular and Cellular Biology:

Nitrogen is essential for fungal growth because it is a component of both nucleic acids and proteins. Fungi have two predominant mechanisms to incorporate ammonium into their metabolism: 1) the NADP-dependent, glutamate-dehydrogenase-catalyzed reductive amination of 2-oxoglutarate to form glutamate; and 2) the ATP-dependent, glutamine synthase-catalyzed fusion of ammonium and glutamate to form glutamine. Beside ammonium, fungi can also utilize a broad variety of other nitrogen sources, such as nitrate, proteins, amino acids, uric acid, allantoin and urea. Efficient control mechanisms are needed to coordinate activation/repression of genes and their products that are involved in sensing, transporting and/or metabolizing nitrogen-containing substances. Furthermore, nitrogen availability plays a critical role in how fungi interact with plants as pathogens and endophytes. Thus, nitrogen limitation has been proposed to be a key signal for activating the expression of virulence-associated genes in plant pathogens. Additionally, quality and quantity of nitrogen also affects the formation of a broad range of secondary metabolites. These secondary metabolites often contribute to virulence on the fungus' host and additionally can bare a threat to animal and human health when they occur as contaminants of food and feed. This Chapter will review the genetic basis of the nitrogen regulation network with the focus on the genus Fusarium which contains some of the most devastating plant pathogens.

Further reading: Fusarium: Genomics, Molecular and Cellular Biology

"There are only a few publications on systems biology studies of infectious processes and no summary literature. In this respect, this book is very timely. It presents a comprehensive analysis of all the components and processes of an infectious process, including applied Omic technologies and biochemical analysis, described in the book in detail. The focus is on the adaptation of the pathogen to the host or in the case of the Y. pestis to life in the flea and the response of host cells to infection with Yersinia. A successful and easy-to-understand book, it presents an overview of the latest findings on the biology and control systems of Yersinia ... it is a must for Yersinia researchers and rewarding for all infection specialists." from Petra Dersch (Braunschweig, Germany) writing in Biospectrum (2013) 19: 224. read more ...

Yersinia: Systems Biology and Control Edited by: Elisabeth Carniel and B. Joseph Hinnebusch ISBN: 978-1-908230-05-8 Publisher: Caister Academic Press Publication Date: July 2012 Cover: hardback "a must for Yersinia researchers" (Biospektrum)

"The literature on this area of ​​research is remarkable and confusing for the non-specialist. This volume edited by Roy Gross and Dagmar Beier is a clear landmark. A top-class science, international team of authors presents in 18 chapters the latest developments in the field of two-component regulatory systems, from genetics through the biochemistry and cell biology to structural biology. I have read with great profit the excellently written and carefully edited contributions. I recommend this book to every student and senior scientist who is interested in approaches to signal transduction processes in microorganisms. The editors and authors have presented the current state of research on a complex topic coherently and for a wide audience." from Erhard Bremer (Marburg, Germany) writing in Biospectrum (2013) 19: 224. read more ...

Two-Component Systems in Bacteria Edited by: Roy Gross and Dagmar Beier ISBN: 978-1-908230-08-9 Publisher: Caister Academic Press Publication Date: August 2012 Cover: hardback "I recommend this book to every student and senior scientist" (Biospektrum)

from Melvyn Smith writing in Real-Time PCR: Advanced Technologies and Applications:

The real-time polymerase chain reaction is now established as one of the core technologies for diagnosing infectious diseases. The early stages of the technique's development were followed by a dramatic increase in the number of diagnostic assays being published, together with the introduction of commercially produced tests. Each of the numerous publications showed a number of differences in the approach to validating the newly-produced assays and in the quality and quantity of the data supporting their validation. As a result, many workers have, at times, found it difficult to reproduce the published results from other laboratories. These difficulties can arise from e.g. a lack of information in the publication, differences in equipment between laboratories, the use of different extraction methods and sequence variations in the pathogen being detected. Over the years a number of authors have voiced their concerns over the subject of what constitutes a properly validated assay, highlighting the issues of basic scientific good practice and the responsibilities of journals in publishing full validation data. This chapter summarises the recent work covering validation and verification methodology in order to provide a practical guide to help inform and standardise the process.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Alan McNally writing in Real-Time PCR: Advanced Technologies and Applications:

The detection and diagnosis of veterinary infectious diseases is an area in which the potential of Real-time PCR has been best demonstrated. In particular Real-time PCR has been successfully applied as a front line tool in the diagnostic algorithm for notifiable veterinary viral pathogens such as Avian Influenza, foot-and-mouth disease, bluetongue virus, as well as rabies and Newcastle disease virus. The rapidly transmissible nature of these agents necessitates near real-time detection and diagnosis in suspected infected animals to allow implementation of control procedures. This chapter will highlight the importance of Real-time PCR in facilitating this rapid diagnosis, and the effect such rapid detection has had on containing and controlling veterinary infectious disease outbreaks.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Patricia Elízaquível, Gloria Sánchez and Rosa Aznar writing in Real-Time PCR in Food Science: Current Technology and Applications:

Foodborne diarrheagenic Escherichia coli strains belong to a minor number of O:H serotypes. Of them the shiga toxin-producing E. coli O157:H7 is the most frequently reported. Besides, non-O157 strains are increasingly being isolated from a variety of food products. E. coli infections are typically associated with transmission through animal products. However, in the last years, contaminated fresh produce is increasingly being implicated in E. coli O157:H7 outbreaks. Currently real-time PCR (qPCR) is considered as an alternative to standard culture methods for E. coli detection in food due to its high speed, specificity, sensitivity, reproducibility and minimization of cross-contamination. Moreover, quantification is possible when an enrichment step is omitted. Although qPCR is a very promising technique for pathogen detection in food, food laboratories and industries are still reluctant to extensively apply it. Real-time qPCR presents some challenges when applying in food, e.g. presence of inhibitors, low levels of cells, detection of dead cells. Besides, the selection of appropriate target regions is another challenge in E. coli detection because of their high genetic heterogeneity. In this review the different approaches proposed to circumvent the difficulties to detect pathogenic E. coli in food and the most frequently targeted genes are presented.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

"The volume's authors are a nice mix of the Neurospora old guard and both relatively new or recently independent members of the Neurospora community. This book will be useful for novice and experienced researchers alike, a go-to manual for graduate students and post-docs new to the field and the organism and any scientist interested in the fungi. It will serve as an entre into less laboratory-amenable but more biologically and medically important fungi ... This terrific new book was a pleasure to read; it belongs in every lab that works on fungi and every academic library. " from Jennifer Loros (The Audrey and Theodor Geisel School of Medicine at Dartmouth Hanover, USA) writing in Curr. Issues Mol. Biol. (2014) 16: 7-8. read more ...

Neurospora: Genomics and Molecular Biology Edited by: Durgadas P. Kasbekar and Kevin McCluskey ISBN: 978-1-908230-12-6 Publisher: Caister Academic Press Publication Date: January 2013 Cover: hardback "a pleasure to read ... belongs in every lab that works on fungi and every academic library" (CIMB)

from Kathie Grant and Corinne Amar writing in Real-Time PCR in Food Science: Current Technology and Applications:

The principle Clostridal foodborne pathogens, Clostridium botulinum and Clostridium perfringens are responsible, respectively, for two different toxin mediated foodborne diseases, namely botulism and C. perfringens food poisoning. Foodborne botulism is a severe, life-threatening disease which can affect a large number of people and although incidence is rare, it is considered a public health emergency. Whilst C. perfringens type A food poisoning is far less severe, it is one of the most common causes of bacterial food poisoning in both the UK and US. It is important to have rapid, accurate methods to detect these two clostridial pathogens and their toxins in order to confirm the cause of illness and identify the food source so that appropriate control and preventative interventions can be implemented. However, conventional laboratory methods to detect C. botulinum and C. perfringens in foods and clinical samples are lengthy, complex, may involve the use of animals and are not always very informative. Real-time PCR assays have been developed to rapidly detect the toxin genes of both pathogens and have been used, in conjunction with culture techniques, to: improve the diagnostic procedure; enhance incident and outbreak investigations and provide information on the pathogenicity of isolates. Real-time PCR detection assays for clostridial foodborne pathogens are also highly valuable to food producers providing faster methods for monitoring growth in food enabling the safety of food products to be assessed more rapidly and effectively. The reliability of real-time PCR detection assays depends upon a range of factors from the bacterial pathogen being detected and the sample matrix to the effective use of controls to ensure the efficiency of the nucleic acid extraction and accuracy of the amplification procedure. This review focuses on the practical application of real-time PCR detection assays for these two clostridial foodborne pathogens.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from George D. Di Giovanni, Gregory D. Sturbaum, and Huw V. Smith writing in Real-Time PCR in Food Science: Current Technology and Applications:

Many parasites are capable of infecting humans, with zoonotic and environmental transmission pathways having significant roles. Of particular significance are pathways involving contaminated food and water. Despite increasing risks posed by food and waterborne parasites due to global sourcing of food, cosmopolitan eating habits, and increased international travel; detection and epidemiologic methods for many of these parasites remains underdeveloped. Microscopy-based detection and diagnostic techniques are still revered as the gold standard for the detection of many food and waterborne parasites. However, the advent and employment of molecular methodologies has proven to surpass microscopy in three major aspects: sensitivity, specificity and the ability to speciate. While molecular methodologies have clear advantages over many traditional detection techniques, standardized PCR methods for the detection of food and waterborne parasites are lacking. This is largely due to multiple obstacles, such as: the diversity of test matrices (e.g. fruits, vegetables, meat products, shellfish, and water); different approaches needed for recovery, concentration, and DNA extraction for different parasites; intrinsically low levels of parasites present in samples; and a lack of multi-laboratory validation of promising methods. Although the PCR detection of food and waterborne parasites may be complex and challenging, recent advances in sample processing techniques and the development of real-time PCR assays are bringing the goal of standardized methodology within view. This review covers an overview of some important food and waterborne parasites, a description of conventional detection methodology, and advances in sample processing and real-time PCR assays. Research needs are discussed along with the benefits of real-time PCR detection and typing.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Doris Helen D'Souza, Marta Hernández, Nigel Cook and David Rodríguez-Lázaro writing in Real-Time PCR in Food Science: Current Technology and Applications:

Analysis of foodstuffs for virus contamination requires profoundly sensitive and accurate methods, due to the potentially low number of viruses and the complexity of the sample matrix. In view of these criteria, the polymerase chain reaction is the assay type of choice, with its rapidity being another useful factor. Real-time PCR (qPCR) is superceding conventional PCR in several areas of molecular diagnostics, and a large variety of published qPCR-based methods for foodborne pathogen detection is available in the scientific literature. In common with other molecular-based methods, qPCR-based analysis of foodstuffs for viruses requires effective controls to ensure that issues associated with low virus numbers and the complexity of the matrix do not result in false negative or positive interpretations of results. These controls are essential for implementation of qPCR-based methods for foodborne virus detection, but in most cases are not included in those which have been published hitherto. Alternative molecular techniques, such as nucleic acid sequence-based amplification (NASBA) and loop-mediated amplification (LAMP) are also suitable for utilization in detection methods for viruses in foods, the same requirements regarding controls pertaining. All molecular-based methods for foodborne virus detection must of necessity contain sample treatment procedures to extract the virus or its nucleic acid out of the food matrix, and these procedures can be elaborate due to matrix complexity. Nonetheless efficient sample treatment methods have been devised, and in combination with molecular assays effective methods for virus analysis are now available for foods. Implementation of these methods in routine diagnostics will support food safety management programs and assist in outbreak investigation, and help to ensure a safe food supply.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

RNA Editing: Current Research and Future Trends Edited by: Stefan Maas ISBN: 978-1-908230-23-2 Publisher: Caister Academic Press Publication Date: June 2013 Cover: hardback

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from Arne Holst-Jensen writing in Real-Time PCR in Food Science: Current Technology and Applications:

Genetic modification (GM) alters the phenotype of the GM organism (GMO). This is achieved through application of gene technology and modification of genetic information stored in nucleic acids. The logical choice of methodology to detect and characterise GM is therefore analytical methods targeting nucleic acids. The polymerase chain reaction (PCR) methodology has been the preferred methodology of this type for two decades, and the following paper will review its applications and derivatives in relation to detection and characterisation of GM organisms (GMOs). The need for detection, identification, characterization and quantitation of GMOs depends on issues such as the legal status of the GMOs in question (authorized or not), labeling or contractual requirements, authentication, traceability and co-existence, environmental monitoring and risk assessments. The fitness for purpose of a specific analytical method is often limited to certain applications. Guidelines to establishment of analytical strategy and method selection can be very useful to those who order as well as to those who provide GMO analyses. A fundamental distinction can be made between screening and identification methods, respectively. The former may be used to group and separate putatively GMO-free samples from samples containing GMO. Both classes of methods may provide qualitative and quantitative information, but only the identification methods can provide accurate quantitation. GMO quantification is achieved almost exclusively with real-time PCR methods, but other alternatives are also available. PCR is also commonly used in combination with other techniques such as Southern blot analyses and DNA sequencing to characterize the genetic constitution of GMOs. Over the last decade extensive resources have been put into validation and critical assessment of performance characteristics and requirements for real-time PCR based GMO detection methods. GMO analyses can be particularly challenging because quantitation is required at very low concentrations, in products of highly variable nature, and where the introduced novel sequences of different GMOs belonging to the same or different species may result in misinterpretation and analytical interference. Consequently, there is a lot to learn from this field of science also for others working with real-time PCR methods. This review will provide several examples.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Carmen Diaz-Amigo and Bert Popping writing in Real-Time PCR in Food Science: Current Technology and Applications:

Food allergens, responsible for IgE-mediated allergic responses and listed in European, North American and Japanese regulations, are exclusively proteins and are ideally detected by analytical methods targeting either peptides or proteins. However, in some cases where no suitable methods for proteins exist or as an alternative method to substantiate results from protein-based methods, DNA-targeting methods can be used as indicators of the presence of potentially allergenic proteins. The advantage of DNA-targeting methods like PCR, real-time PCR is presently the lower cost and availability of free literature on several detection systems, including a certain degree of multiplexing. Clear disadvantages include the poor sensitivity for egg, milk and samples containing inhibitors (like polyphenols in chocolate) as well as its limited applicability in some industrial protein concentrates. In addition, if quantitative results need to be obtained, the DNA-based system needs to be calibrated for each matrix tested as protein-to-DNA composition is typically matrix specific. However, PCR based methods are well established in many laboratories and still regularly used. This review discusses suitable systems for detection of DNA of ingredients and foods containing allergenic proteins, potential pitfalls and multiplex capabilities of such systems.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Ann B. Hill writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

In recent years it has been suggested that CMV may be involved in the pathogenesis of a variety of conditions in which there may not be clear evidence of viral replication. These "non-traditional" disease associations include glioblastoma and various other cancers, atherosclerotic cardiovascular disease, Alzheimer's disease, and immunosenescence, amongst others. The pathologies fall into two broad groups: tumors, and inflammatory diseases of aging. In the case of tumors, some groups have used ultrasensitive detection techniques and report finding CMV in the majority of tumor cells. In the case of inflammatory diseases of aging, the evidence mostly comes from epidemiological studies that have associated CMV serology or CMV-driven alterations in T cell populations with various outcomes. While CMV's biology provides ready explanations for these putative disease associations, the actual evidence for its being involved remains controversial. This review will review the evidence for several putative disease associations.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention

from Keith Poole writing in Microbial Efflux Pumps: Current Research:

Antibiotic efflux systems are common in Pseudomonas aeruginosa, with chromosomally-encoded multidrug efflux systems of the Resistance Nodulation Division (RND) family, specifically MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, of particular importance in clinical settings. Despite the broad substrate specificity of many of these, their clinical importance is limited to fluoroquinolone resistance (MexAB-OprM, MexCD-OprJ and MexEF-OprN), β-lactam resistance (MexAB-OprM, MexXY-OprM) and aminoglycoside resistance (MexXY-OprM). Expression of these systems is governed by the products of regulatory genes (mexAB-oprM: mexR, nalC, nalD; mexCD-oprJ: nfxB; mexEF-oprN: mexT; mexXY: mexZ) whose mutation is typically responsible for acquired multidrug resistance in lab and clinical isolates. With few exceptions these efflux systems are not inducible by substrate antimicrobials, consistent with antimicrobial efflux not being their intended function. Indeed, recent data highlight their induction by environmental stresses (oxidative stress, nitrosative stress, envelope stress) suggestive of a role in stress response systems in this organism. Significantly, such stresses may provide a selective pressure for antibiotic-resistant efflux mutants in vivo independent of antibiotic exposure. Given the importance of these efflux systems in intrinsic and acquired multidrug resistance in P. aeruginosa, strategies aimed at interfering with efflux-mediated resistance are being investigated.

Further reading: Microbial Efflux Pumps: Current Research

from Brandon L. Coyle, Weibin Zhou and François Baneyx writing in Bionanotechnology: Biological Self-assembly and its Applications:

Designer proteins combine the adhesive or synthesizing properties of solid binding peptides (SBPs) selected by combinatorial techniques with the desirable characteristics of a host scaffold. Like natural biomineralizing proteins, these chimeric constructs are powerful tools to control the nucleation, growth, morphogenesis and crystallography of inorganic phases. They also hold great potential for the assembly of hybrid structures in which inorganic, biological and synthetic components are organized with the high degree of precision needed to take advantage of the unique properties of matter at the nanoscale. After briefly discussing common approaches for identifying SBPs, we discuss the mechanisms by which they modulate materialization, which variables influence the process, and review recent progress in the use of designer proteins to fabricate complex architectures.

Further reading: Bionanotechnology: Biological Self-assembly and its Applications

from Michael Mach, Anna-Katharina Wiegers, Nadja Spindler and Thomas Winkler writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

The generation of antibodies represents a powerful tool of the adaptive immune system in the battle against viral infections. Targets for antibodies with potential antiviral activity are glycoproteins in the viral envelope and/or on the surface of infected cells. In recent years, considerable progress has been made in our understanding of the protective antibody response against cytomegaloviruses. Animal studies have unambiguously demonstrated the protective capacity of antibodies both in prophylaxis as well as in therapy of existing primary infection or reactivation. A number of human monoclonal antibodies have been isolated which show potent virus-neutralizing capacity and new antibody targets have been identified. However, we still need to expand our knowledge on the mechanisms of virus neutralization by antibodies and the mode of action of protective antibodies in vivo. Increasing this knowledge will help us to rationally design strategies to limit the consequences of infections in populations at risk for CMV-disease.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention

from Christof Godts, Gitte Loozen, Marc Quirynen and Wim Teughels writing in Oral Microbial Ecology: Current Research and New Perspectives:

The human oral cavity is colonized by a wide variety of bacteria, which form very complex and dynamic biofilms on hard and soft tissues. Certain members of these microbiological communities are associated with oral infections, such as caries and periodontal diseases. New treatment approaches are emerging that do not rely on conventional antibiotic therapies, since complete eradication of pathogenic bacteria from oral biofilms is impossible and antibiotic resistance is becoming problematic. For example, attempts have been made to reduce the overall pathogenicity of tissue-associated biofilms by introducing live beneficial bacteria. Early successes, primarily in the field of gastro-intestinal microbiology, have paved the way for the introduction of probiotics in oral health care. These new anti-/pro-microbial therapies are considered very promising for prevention and treatment of plaque related oral diseases. In this review, the concept of probiotics for oral healthcare is introduced, followed by an overview of the diverse mechanisms of probiotic action in the oral cavity. Since the benefits of probiotics will ultimately be revealed by clinical studies, the clinical outcomes of probiotic applications for combating dental caries and periodontal diseases are addressed. Finally the interactions of probiotics with the oral microbial ecosystem are discussed and future perspectives regarding the oral probiotic concept are presented.

Further reading: Oral Microbial Ecology: Current Research and New Perspectives

from Robert F. Kalejta writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

As virions disassemble during viral entry, they must expertly navigate and manage the complex and unwelcoming environments they encounter in order to successfully infect host cells. Herpesviruses incorporate proteins into their virions in a layer between the capsid and envelope termed the tegument to assist in this hostile takeover. When delivered to infected cells subsequent to membrane fusion, tegument proteins begin to facilitate viral infection after entry but before immediate early (IE) gene expression (referred to as the pre-IE stage of infection). Tegument-delivered proteins mediate capsid migration through the cytoplasm to nuclear pore complexes and the transmission of the genome into the nucleus. Furthermore, they modulate viral transcription, and help infected cells avoid all three classes of immune function (intrinsic, innate and adaptive). While they are most often studied during lytic infections, a new appreciation for the role that the proper regulation of tegument-delivered protein function may play during viral latency is emerging. Here the pre-IE functions of tegument proteins during both lytic and latent infections are reviewed and analyzed.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention

Microbial Efflux Pumps: Current Research Edited by: Edward W. Yu, Qijing Zhang and Melissa H. Brown ISBN: 978-1-908230-21-8 Publisher: Caister Academic Press Publication Date: June 2013 Cover: hardback

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from Jenny Draper, Jinping Du, David O. Hooks, Jason Lee, Natalie Parlane and Bernd H.A. Rehm writing in Bionanotechnology: Biological Self-assembly and its Applications:

Biopolyesters are a class of carbon storage polymers synthesized by a wide variety of bacteria in response to nutrient stress. Production of these polyhydroxyalkanoates (PHAs = polyesters) is catalyzed by PHA synthases, which polymerize (R)-3-hydroxyacyl-CoA thioesters into polyester. There are several different classes of PHA synthases which preferentially utilize different CoA thioester precursors, generating PHAs with varying material properties such as elasticity and melting point. Genetic engineering and growth on varied carbon sources can be used to modify the type of polyester produced. The general biopolyester properties of biocompatibility, biodegradability, and production from renewable carbon sources have led to considerable interest in PHAs as biomaterials for medical applications as well as alternatives to petrochemical plastics. Biopolyesters are generated in the cell as water-insoluble granules coated with structural, regulatory, and synthase proteins. Recently, the natural structure of the granules has been exploited to generate functionalized nanoparticles for use in a wide variety of applications, including bioseparation, drug delivery, protein purification, enzyme immobilization, diagnostics, and vaccine delivery.

Further reading: Bionanotechnology: Biological Self-assembly and its Applications

from Jane M. Carlton, Steven A. Sullivan and Karine G. Le Roch writing in Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology:

It may be a cliché to state, but obtaining the genome sequence of an organism is one of the most important – if not the most important – step towards interrogating its biology. The first two malaria parasite genome sequences (Plasmodium falciparum and the rodent model Plasmodium yoelii yoelii) were published in 2002 after more than half a decade of intense sequencing, assembly gap closure, and sequence annotation. Since then, reference genomes of several more Plasmodium species have been generated, with an emphasis on malaria parasites that infect humans due to their global health importance. With the recent transformation in technologies available for the rapid and cheap production of genome sequence data, an explosion of P. falciparum genomes from a wide variety of geographical locations has started to appear, and with it all of the computational issues of large dataset manipulation, storage and analysis. We begin this chapter with a discussion of sequencing technologies, from Sanger sequencing through to current next generation sequencing platforms, to lay the foundation for many of the studies that are presented in this book. Next we describe the characteristics of a typical Plasmodium nuclear genome (with reference to those species of malaria that infect mammals), with a brief mention of the extranuclear apicoplast and mitochondrial genomes also found in the parasite. Finally, we outline how comparative genomics - literally comparing genomes within and between species - has been used as a powerful tool to elucidate malaria parasite biology and evolution..

Further reading: Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology

Cytomegaloviruses: From Molecular Pathogenesis to Intervention Edited by: Matthias J. Reddehase ISBN: 978-1-908230-18-8 (Two Volume Box Set) Publisher: Caister Academic Press Publication Date: April 2013 Cover: hardback

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from Lisa C. Ranford-Cartwright, Karen l. Hayton and Michael T. Ferdig writing in Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology:

Experimental genetic crosses mimic the sexual reproduction process, and accompanying genetic recombination, that occurs between individuals of the same species during natural transmission. Experimental crosses performed using rodent and human species of Plasmodium have been used to link phenotype and genotype for a variety of traits, and have been particularly useful for understanding phenotypes for which no obvious candidate genes are known. In addition, analysis of experimental crosses has provided insights into the frequency and types of recombination that occur. Some biological traits are explained by inheritance of single genes, whereas several loci, known as quantitative trait loci (qtl), contribute to a "complex trait". Linkage analysis of experimental genetic crosses of Plasmodium falciparum have identified parasite loci contributing to resistance to a number of antimalarial drugs such as chloroquine and quinine, as well as loci controlling the ability of parasites to invade erythrocytes of different primate species, the ability to infect mosquitoes, and intraerythrocytic growth rates. Genetic mapping can also identify genomic regions associated with the control of gene expression (expression qtl or eQTL). Genetic crosses and genetic mapping continue to play a significant role in our understanding of malaria parasite biology, transmission, and drug resistance.

Further reading: Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology

"timely and critical appraisal ... for PhD students and researchers working with malaria parasites, this text represents an essential and eminently accessible resource for their work." from Paul Horrocks (Keele University Medical School, UK) writing in Parasites and Vectors (2013) 6: 74. read more ...

Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology Edited by: Jane M. Carlton, Susan L. Perkins and Kirk W. Deitsch ISBN: 978-1-908230-07-2 Publisher: Caister Academic Press Publication Date: February 2013 Cover: hardback "an essential and eminently accessible resource" (Parasites and Vectors)

from Anisha David, Sunita Yadav and Satish Chander Bhatla writing in Bionanotechnology: Biological Self-assembly and its Applications:

Although oil bodies are present in a wide variety of tissues in plants, it is their abundance in the oilseed cotyledons that has been most extensively investigated for their biogenesis, structure, physiological roles, isolation and biotechnological applications. The phospholipid monolayer membrane of the oil bodies encasing the triacylglycerol (TAG) matrix not only possesses a set of structural and functional proteins (oleosins, steroleosins and caleosins), they also exhibit quite a few enzymatic and non-enzymatic proteins on their surface (lipoxygenase, protease and phospholipase) whose expression is transient and depends on the stage of oil body mobilization during seed germination. These transiently expressed signalling molecules are under the influence of various environmental and consequent physiological factors for their roles in oil body mobilization during seed germination. Based on these features of oil bodies to attract and bind a variety of biomolecules on their surface, oil body preparations have been put to extensive biotechnological applications, which are also being discussed in this review.

Further reading: Bionanotechnology: Biological Self-assembly and its Applications

from George Hajishengallis writing in Oral Microbial Ecology: Current Research and New Perspectives:

The polymicrobial community that initiates periodontal disease does not represent a random compilation of bacteria. Rather, these bacteria form organized consortia that have evolved through mutually beneficial relationships. This review focuses on microbial immune subversion as a means by which select pathogens may contribute to the adaptive fitness of the entire periodontal biofilm. For instance, Porphyromonas gingivalis expresses specialized virulence traits that undermine immunity and promote non-resolving inflammation, which, respectively, protect the bacteria and facilitate nutrient acquisition. The virulence factors involved (e.g. cysteine proteases and atypical lipopolysaccharide structures) are released as components of readily diffusible membrane vesicles, which can thus become available for the benefit of other biofilm organisms. The elucidation of immune subversion mechanisms of key periopathogens that promote the collective virulence of their communities may provide new avenues of therapeutic intervention in human periodontitis.

Further reading: Oral Microbial Ecology: Current Research and New Perspectives

from John G. Thomas writing in Oral Microbial Ecology: Current Research and New Perspectives:

The link between oral flora and lung infections in mechanically ventilated (MV) patients (the 'Oral Systemic Link') has always been circumstantial, based on clinical and nursing practices and preventative care. At the turn of the 20th century, that began to change as molecular and imaging methods provided tools to better evaluate microbial exchange, refocusing on the importance of the endotracheal tube (ETT) lumen as a potential conduit, devoid of normal cellular defensive components. We undertook the challenge in two phases, first engineering the Adult -Ventilator Endotracheal Lung (A-VEL) simulator to replicate the closed, bi-directional airway and stress of the intubated patient in the intensive care unit (ICU). Of singular importance, was the incorporation of multiple quantitative imaging techniques to define the 3-D biofilm luminal development in Stages (I-IV) from single to complex microbial communities and the incorporation of dental pathogens (Streptococcus mutans and Porphyromonas gingivalis) as an endogenous event, in the preconditioning of the ETT luminal surface, an abiotic medical device. The second phase shifted to the in vivo environment and in multiple clinical studies, unmasked the bi-phasic nature of ETT luminal colonization, oral-endogenous (Early) to systemic - exogenous (Late) at a 3-5 day 'switch'. Further, the ICU studies dramatized the shift from the infectious process in VAP to Work of Breathing (WOB), where the former occurred in 16%, while biofilm accretion, occlusion of the ETT lumen and increased airway resistance occurred in 100% of intubated patients. Most recently, we have used both 16S (microarray) and 18S rRNA (pyrosequencing) to redefine the proportions of bacteria and fungi from oral reservoirs, and been astonished by the richness and diversity of the oral fungal community in the ETT accretion occlusion, often yielding >15 species . Endotracheal Tube Associated Pneumonia (EAP) management continues to elude optimal strategies, but the use of selected oral probiotics coupled with better oral care in both the ICU and admitting institutions is gaining reinforcement: "Oral Stewardship". Further, the utilization of dental professionals in the ICU has importance, as has the recognition that the next fertile area of airway disease (oral to systemic) study is the neonatal intensive care unit (NICU), where 50% of newborns may be intubated and develop EAP with no teeth. How? Why?

Further reading: Oral Microbial Ecology: Current Research and New Perspectives

from Marieke P.T. Otten, Henk J. Busscher, Chris G. van Hoogmoed, Frank Abbas and Henny C. van der Mei writing in Oral Microbial Ecology: Current Research and New Perspectives:

An overview is presented on oral biofilm formation and recent developments in oral biofilm control using mechanical devices (manual or powered toothbrushes and interdental cleaning devices) and biofilm control based on oral chemotherapeutics (antibacterial toothpastes and mouthrinses). For clinical efficacy of oral chemotherapeutics, it is important that the antibacterial remains active in the oral cavity for periods longer than the actual brushing or rinsing time, a characteristic called 'substantivity'. Substantivity can be achieved by adsorption of antibacterials to oral hard and soft tissues followed by release. Mechanical cleaning never results in complete removal of oral biofilm: most notably in fissures, interproximal spaces, gingival pockets and around orthodontic appliances. Recently, it has been demonstrated that this residual biofilm can act as a reservoir for oral chemotherapeutics that are slowly released over time in bio-active concentrations. This function of oral biofilm was already known for fluoride and has been demonstrated to aid in preventing caries. Results from our laboratory showed that residual biofilm after mechanical cleaning can release absorbed antibacterial agents from toothpastes and mouthrinses in bio-active concentrations.

Further reading: Oral Microbial Ecology: Current Research and New Perspectives

from Sarath Chandra Janga and Gabriel Moreno-Hagelsieb writing in Bacterial Gene Regulation and Transcriptional Networks:

An average of 60% of prokaryotic genes are organized into operons-polycistronic transcription units, making them a very important feature of their genomic organization. Operons most commonly contain genes whose products have functional associations and are abundant because they constitute an easy means for coregulation and the associated genes can act as a functional unit with a higher success rate in horizontal gene transfer events than single genes. Operons are transcribed from a single promoter, thus rarely needing genomic features between their constituting genes, naturally resulting in shorter distances between genes in operons than between adjacent genes in different transcription units. Thus, operons can be predicted based on distances between adjacent genes in the same DNA strand. This feature, intergenic distance, is the most informative criterion for predicting operons. However, predictions based on conservation of gene order followed by phylogenetic profiles, provide cleaner predictions, albeit with much lower coverage. Transcriptional terminators and other sequence features might add quality to operon predictions, but the gain is minimal for most prokaryotes. Operon organization is not well conserved with evolutionary divergence. However, operons rearrange in a functionally coherent manner. Thus, the combination of operon predictions with operon rearrangements constitutes the most powerful source for the prediction of functional associations by genomic context in prokaryotes.

Further reading: Bacterial Gene Regulation and Transcriptional Networks

from Agustino Martínez-Antonio writing in Bacterial Gene Regulation and Transcriptional Networks:

Transcription factors function as sensory systems acting at the core of genetic regulatory switches. The transcriptional regulatory network in Escherichia coli can be studied as the integration of the whole of these genetic sensory systems. The operation of this regulatory system affect the expression of genes by interacting with the DNA at the promoter regions of transcription units. In this review I present the advances of what we know about the mechanistic logic for the operation of the regulatory program in E. coli. It is proposed that for a better understanding on the operation of the regulatory network it should be considered the globalism of transcription factors, the signal perceived by each, their co-regulating activity, the genome position of regulatory and target genes, and cellular concentration of the regulatory proteins, among others.

Further reading: Bacterial Gene Regulation and Transcriptional Networks

from Klaus Früh, Daniel Malouli, Kristie L. Oxford and Peter A. Barry writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

The last few years have witnessed significant expansion of the Non-Human-Primate (NHP) models of CMV persistence and pathogenesis. Progress in the utilization of the NHP CMV models has been highlighted by a better understanding of natural history, comparative genomic sequence analyses, and in vivo studies addressing mechanisms of tissue tropism, immune modulation, vaccine development, and optimization of the use of CMV as a vaccine vector for ectopic expression of heterologous antigens. The earliest observations of CMV infection in NHP during the first part of the twentieth century were remarkable for their prescient descriptions of CMV-host relationships based entirely on microscopic characterization of the protozoan-like (cytomegalic) cells that had been noted in congenitally infected human infants (Ribbert, 1904; Goodpasture and Talbot, 1921) and guinea pigs (Jackson, 1920). In particular, it was noted in the 1920's and 1930's that NHP CMV (1) is a ubiquitous infectious agent, (2) infects multiple cell types, (3) is characterized by low virulence, and (4) modifies host inflammatory responses. In addition, the first use of the term "latency" to describe the ability of CMV to reactivate may have been used for NHP CMV. In 1935, Cowdry and Scott recognized that treatment of CMV-infected monkeys with irradiated ergosterol stimulated reactivation of CMV in multiple tissues, and they noted that the treatment "may have activated or intensified a process already latent in the kidneys" (Cowdry and Scott, 1935). The recent progress in the NHP models follows these earliest insights into the hallmarks of CMV infections, and now enables the unique positioning of NHP models to provide a better understanding, treatment, and prevention of HCMV infection and disease in humans. This review summarizes the current status of our understanding of NHP CMVs with particular emphasis on viral gene function and viral disease models.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention

from Gavin W. G. Wilkinson, Rebecca J. Aicheler and Eddie C. Y. Wang writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

The efficient downregulation of HLA-I by HCMV has the clear potential to render infected cells extremely vulnerable to NK cells. Moreover, the major IE genes activate cell responses that stimulate efficient transcription of multiple ligands for NK cell activating receptors. The capacity of HCMV to persist in vivo can clearly be ascribed to its ability to modulate NK cell responses. To date 7 functions encoded by HCMV have been formally demonstrated to suppress NK cell activation. UL18 is an MHC-I homologue that binds the inhibitory receptor LIR-1, while UL40 rescues expression of HLA-E, a ligand for the inhibitory receptor CD94-NKG2A. UL16, UL142 and miR-UL112 target multiple ligands for the ubiquitous NK activating receptor NKG2D, while UL141 targets ligands for the ubiquitous activating receptors DNAM-1 and TACTILE. The UL83-encoded major tegument protein (pp65) is unique in that it binds directly to inhibit the activating receptor NKp30. It is becoming evident that a substantial proportion of the remarkable coding capacity of this virus is directed at systematically addressing the NK cell response. Outwith the immediate goal of understanding HCMV pathogenesis, research on these immunodulatory functions are providing remarkable insights into the mechanisms that regulate human NK cell responses. Recent studies demonstrate that during its lifelong persistent/latent infection, HCMV induces dramatic changes in the NK cell repertoire leading specifically to expansions of NK cell subsets expressing CD94-NKG2C, LIR-1 and CD57. There is growing interest in these changes in the NK cell response as they potentially contribute to an emerging paradox: an 'adaptive' response by a supposedly innate arm of the immune system. This amplification of specific NK cell subsets to the virus may be instrumental in controlling infections, and may also be disrupted by immunosuppression. NK cells undoubtedly play a crucial role in controlling HCMV infections. There is a compelling need to understand the mechanisms by which HCMV evades, modulates, and ultimately is recognised by 'innate' defence systems.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention

from Lisa P. Daley-Bauer and Edward S. Mocarski writing in Cytomegaloviruses: From Molecular Pathogenesis to Intervention:

Cytomegalovirus pathogenesis, dissemination and immunity are tied to the behaviour of myelomonocytic cells. Investigations of the roles of monocyte subsets in the dissemination of cytomegalovirus revealed that the MCMV-encoded chemokine, MCK2, controls recruitment patterns of the two major monocyte subsets (inflammatory and patrolling) to sites of infection. Monocytes give rise to both macrophages and dendritic cells that populate tissues. Mice deficient in the chemokine axis (CCR2 and CCL2 /MCP-1) do not support inflammatory monocyte emigration from bone marrow. Inflammatory monocyte-derived lineages, which are nonpermissive for MCMV, are dispensable for pathogenesis and dissemination as well as for the establishment of latency set-points. Nevertheless, recruitment of inflammatory monocytes is enhanced by elaboration of MCK2 and impairs the CTL response to delay viral clearance. In contrast, patrolling monocytes support MCMV replication and contribute to dissemination patterns in the host. Studies in CX₃CR1-deficient infected mice show reduced viral dissemination to salivary glands, consistent with the reduced survival of patrolling monocytes in these animals. HCMV studies suggest myelomonocytic progenitor cells, including inflammatory and patrolling monocyte subsets, are associated with acute as well as latent infections. MCMV latency in mice occurs in myeloid as well as epithelial and endothelial cell lineages. In this paper, we review the current understanding of myelomonocytic lineage cells in the establishment of cytomegalovirus infections based on human and murine studies.

Further reading: Cytomegaloviruses: From Molecular Pathogenesis to Intervention