from Karlijn C. Bastiaansen, Wilbert Bitter and María A. Llamas writing in Bacterial Regulatory Networks:

Gene expression in bacteria is mainly controlled at the level of transcription initiation. To achieve this process a number of different mechanisms have evolved, one of which is the utilization of alternative sigma factors. Sigma factors are small proteins that associate with the RNA polymerase core enzyme (RNAPc) and direct it to specific promoter sequences, where they initiate gene transcription. Bacteria are able to regulate transcription initiation by synthesizing and activating different sigma factors that recognize different promoter consensus sequences. The largest group of alternative sigma factors consists of the so-called extracytoplasmic function (ECF) sigma factors that regulate gene expression in response to cell envelope stresses or environmental stimuli. The activity of ECF sigma factors is controlled by anti-sigma factors and a complex cascade of regulated (proteolytic) modifications. In gram-negative bacteria, ECF sigma factors are also controlled by cell-surface signalling (CSS), a regulatory system that includes an outer membrane receptor in the signal transduction pathway. In this chapter we will discuss the general composition and function of ECF sigma factors and their role in cell envelope stress responses and CSS.

Further reading: Bacterial Regulatory Networks   Related publications

from Moshe Szyf writing in Epigenetics: A Reference Manual:

The DNA molecule contains within its chemical structure two layers of information. The DNA sequence that bears the ancestral genetic information and the pattern of distribution of covalently bound methyl groups to cytosines in DNA. While the genetic information is similar in all tissues in the individual, the pattern of distribution of methylation across the genome is cell-type specific. DNA methylation is an important regulator of gene function. Recent data that will be discussed here that supports the hypothesis that DNA methylation is a reversible biological signal. This expands the potential role of DNA methylation beyond embryogenesis to other time-points in life and to post mitotic tissues such as the brain. DNA methylation is proposed to act as a genomic response to both physical and social signals from the environment at different time points in life and to serve as a genomic memory of these exposures at different time scales, stably altering gene expression programming and thus modulating the physical and behavioral phenotypes to respond to these environments. It is hypothesized that DNA methylation provides within the structure of the DNA a dynamic interface between the changing world around us and the relatively fixed and stable genome.

Further reading: Epigenetics: A Reference Manual

from Eivind Almaas, Per Bruheim, Rahmi Lale and Svein Valla writing in Systems Microbiology: Current Topics and Applications:

The functional repertoire of an organism's metabolic network is closely linked to its phenotype and potential for utility in metabolic engineering applications. In this chapter, we discuss a systems biology view of Escherichia coli metabolism by integrating current genome-scale computational modelling approaches with available molecular genetics tools, as well as the experimental framework for metabolite and metabolic flux determination.

Further reading: Systems Microbiology   Related publications

from J. Maxwell Dow, Yvonne McCarthy, Karen O'Donovan, Delphine Caly and Robert P. Ryan writing in Bacterial Regulatory Networks:

Cyclic di-GMP is now recognised as an almost universal second messenger in eubacteria that acts to regulate a wide range of functions including developmental transitions, adhesion, biofilm formation, motility and the synthesis of virulence factors. Cyclic di-GMP is synthesised from two GTP molecules by diguanylate cyclases that have a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. These proteins often have associated signal input domains, suggesting that their enzymatic activity may be modulated by different environmental or cellular cues. Cyclic di-GMP exerts a regulatory action through binding to diverse receptors that include a small protein domain called PilZ, transcription factors, enzymatically-inactive GGDEF, EAL or HD-GYP domains and riboswitches. The multiplicity of GGDEF, EAL and HD-GYP proteins together with a range of receptors within the same bacterial cell indicates the considerable complexity of cyclic di-GMP signalling. This has led to the concept of discrete pools of the nucleotide that are generated locally and act to influence intimately associated targets. A number of signalling proteins may be organised in a regulatory network to control a common function(s). Understanding cyclic di-GMP signalling may afford strategies for inhibition of biofilm formation and virulence factor synthesis in bacterial pathogens.

Further reading: Bacterial Regulatory Networks   Related publications

from John M. Pfeffer, Patrick J. Moynihan, Chelsea A. Clarke, Chris Vandenende and Anthony J. Clarke writing in Bacterial Glycomics: Current Research, Technology and Applications:

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes however, the lytic transglycosylases are not hydrolases, but instead cleave the glycosidic linkage between N-acetylmuramyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramyl product. They are ubiquitous in bacteria which produce a complement of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. Given their catastrophic autolytic potential, the activity of lytic transglycosylases must be tightly controlled within the producing cells. Three modes of control at the enzymatic level have been identified: the modification of substrate, membrane association and complex formation, and the production of proteinaceous inhibitors. These modes of control and their potential as new targets for antibacterials are discussed.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Anne N. Reid and Leslie Cuthbertson writing in Bacterial Glycomics: Current Research, Technology and Applications:

Capsular polysaccharides (CPSs) and exopolysaccharides (EPSs) enhance bacterial survival in the environment, contribute to symbiotic interactions between plants and bacteria, and mediate interactions between plant and animal pathogens and their hosts. Bacteria express a wide array of CPS and EPS structures that are assembled by one of three distinct mechanisms. The Wzy-dependent polymerization system is characterized by the synthesis of lipid-linked repeat units in the cytoplasm, and their block-wise polymerization at the periplasmic face of the inner membrane. The resulting polymer is transported across the outer membrane (in Gram-negative organisms) via a channel formed by an outer membrane polysaccharide export (OPX) protein. The ATP-binding cassette (ABC) transporter-dependent system is defined by the synthesis of full-length CPS chains in the cytoplasm, their ABC transporter-dependent export across the inner membrane, and their subsequent transport across the outer membrane, presumably via a channel formed by an OPX protein. In the synthase-dependent system, a single enzyme achieves polymer initiation, synthesis and export across the membrane. This chapter describes these modes of CPS and EPS assembly, highlighting recent findings and identifying areas where further research is warranted.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from James D. Bryers writing in Microbial Biofilms: Current Research and Applications:

Clinically related research on biofilms has expanded exponentially in the past ten years due to the pandemic of nosocomial (hospital-related) infections. Biofilms are thought to cause a significant amount of all human microbial infections, according to the Centers for Disease Control and Prevention. Nosocomial infections are the fifth leading cause of death in the U.S. with more than two million cases annually (or approximately 10% of American hospital patients). The difficulty of eradicating biofilm bacteria with classic systemic antibiotic treatments is a prime concern of medicine. Biofilm bacteria can be up to a thousand times less susceptible to antimicrobial stress than their freely suspended counterparts. This chapter discusses the pathogenesis of a number of biofilm-mediated infections, including: oral infections, biomedical device based infections, osteomyelitis, otitis media, and others. Emerging research in biofilm control and prevention is also reviewed.

Further reading: Microbial Biofilms: Current Research and Applications

from Patricia C. Burrows, Simone C. Wiesler, Zhensheng Pan, Martin Buck and Sivaramesh Wigneshweraraj writing in Bacterial Regulatory Networks:

Amongst the many accessory factors that bind RNA polymerase (RNAp) and serve to control its activities, sigma (σ) factors ubiquitously feature in programming of gene expression in response to abiotic and biotic cues. Here we review the role of the major variant σ factor, σ⁵⁴, in the expression of gene sets used for establishing the virulence of a wide range of pathogenic bacteria. The tight coupling of σ⁵⁴-dependent transcription to signalling pathways underpins the regulation of such systems, and allows a wide dynamic range of gene expression.

Further reading: Bacterial Regulatory Networks   Related publications

from Arun K. Mishra, Sarah M. Batt, Luke J. Alderwick, Klaus Futterer, and Gurdyal Singh Besra writing in Bacterial Glycomics: Current Research, Technology and Applications:

Lipoarabinomannan is an amphipathic lipoglycan found in the cell wall of most Actinomycetes. The majority of bacteria from the sub-order Corynebacterineae, including Mycobacterium tuberculosis, Mycobacterium smegmatis and Corynebacterium glutamicum, and from genus Rhodococcus, Gordonia and Amycolatopsis; all possess lipoarabinomannan and related glycoconjugates, such as lipomannan and phosphatidyl-myo-inositol mannosides. In addition to their physiological function in these microorganisms, these glycoconjugates play a key immunomodulatory role for pathogenic bacteria during infection. Herein, we report the work from this laboratory and several others, which has led to the biochemical characterization of key enzymes involved in the biogenesis of these complex glycoconjugates.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Susan M. Twine and Susan M. Logan writing in Bacterial Glycomics: Current Research, Technology and Applications:

The biosynthesis and assembly of the flagellar apparatus has been the subject of extensive studies over many decades. More recently, glycosylation of the major structural protein, the flagellin, has been shown to be an important component of numerous flagellar systems in both Archaea and Bacteria, playing either an integral role in assembly and for a number of bacterial pathogens a role in virulence. Increasingly, it is apparent that bacteria elaborate a structurally diverse array of flagellin-modifying glycans. This chapter focuses firstly upon reviewing recent research on the structural diversity in Gram-positive and Gram-negative flagellar glycosylation systems. In the second part, the ways in which flagellin glycosylation and associated biosynthetic pathways can be exploited are discussed.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Kathryn A. Scott, Elizabeth E. Jefferys, Benjamin A. Hall, Mark A. J. Roberts and Judith P. Armitage writing in Bacterial Regulatory Networks:

Chemotaxis is the process by which bacteria migrate towards environments that are favourable for growth. Changes in the concentration of attractants or repellents are detected by receptors, which are usually transmembrane proteins. These receptors transduce the signal to the interior of the cell where a two-component system ultimately leads to changes in motile behaviour. Chemotaxis emerged as a beneficial trait for survival early in the evolution of bacteria and archaea. A core set of proteins is common to the chemosensory networks in many different species. During the evolution of bacteria this core network has diversified and expanded. Here we describe the conserved apparatus in the steps necessary for chemotaxis; sensing of chemoeffectors, signalling to the motility apparatus, rapid signal termination, and adaptation. We then highlight examples from species with complex chemosensory networks to illustrate the variations in chemotactic apparatus that have arisen from the common core.

Further reading: Bacterial Regulatory Networks   Related publications

from Diana Clausznitzer, Judith P. Armitage and Robert G. Endres writing in Systems Microbiology: Current Topics and Applications:

Bacterial chemotaxis is a paradigm for biological sensing and information transmission. The chemotaxis signal-transduction pathway allows cells to sense chemicals in their surroundings in order to regulate flagellated rotary motors, thus allowing them to swim towards nutrients and away from toxins. Importantly, cells are able to sense with remarkably high sensitivity over a wide range of chemical background concentrations. To make this possible, chemoreceptors do not signal independently but form clusters for amplification and integration of signals, as well as for adaptation to persistent stimulation. While chemotaxis in Escherichia coli has been exceptionally well characterised, new experimental facts still require revisions of existing models and thus further increase our understanding of sensing and signalling in bacteria. Additionally, experiments on other bacterial species such as Bacillus subtilis and Rhodobacter sphaeroides indicate that bacteria other than E. coli can have substantially different and more complex chemotaxis pathways, which provides renewed challenges for experimentalists and modellers alike. Here we discuss our current understanding as well as the frontiers of bacterial chemotaxis research.

Further reading: Systems Microbiology   Related publications

from J. Cuccui, R.H. Langdon, M.G. Moule and Brendan W. Wren writing in Bacterial Glycomics: Current Research, Technology and Applications:

Once thought to be restricted to eukaryotes and archaea, N-linked glycosylation has now been discovered in prokarytoes. Over the past decade, our understanding of bacterial N-linked glycosylations systems and their abundance has been expanding. This type of protein modification was first demonstrated in Campylobacter jejuni, a human gut pathogen, and we now know that N-linked glycosylation also exists in other &episilon;-proteobacteria ranging from the deep-sea vent Nitratiruptor spp. and Sulfurovum spp. to sulfate reducing δ-proteobacteria. A greater understanding of these systems is necessary in order to comprehend the evolutionary reasons for their development and maintenance. In addition, this knowledge may also be exploited for glycoengineering purposes to produce cheaper subunit vaccines as well as humanized proteins.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Warren Wakarchuk writing in Bacterial Glycomics: Current Research, Technology and Applications:

It is now accepted that complex glycans play major roles in biology, such as the development of the embryo, the function of the immune system, microbial and viral pathogenesis and cellular communication, to name just a few. The many faceted roles that glycans play in biology makes them a challenge to understand on functional level, and the complexity of the structures themselves makes them daunting targets for chemical synthesis, which is required for examination of their binding interactions and for future development of carbohydrate based therapeutics. In order to facilitate the synthesis of complex glycans, we have been examining glycosyltransferases which make strategic linkages in biologically active glycans. Many of the mammalian enzymes have not been as easy to express as active recombinant proteins, and many have a more restricted acceptor specificity that limits their use for synthesis. Our focus has been on the use of bacterial enzymes from pathogens which make molecular mimics of host glycans, and which have been shown to be potent catalystsfor carbohydrate synthesis. This chapter will provide a review on a variety of bacterial enzymes that we and others have enabled for in vitro synthetic carbohydrate chemistry, as well as some promising in vivo production strategies for bioactive carbohydrates.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Hengmi Cui, Isabelle Cui and Xi Yang writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

Antisense RNA is the first RNA molecule identified to function as a regulator in the cell. With advances in biological science, antisense RNA is now recognized to be involved in not only post-transcriptional regulation, but also the transcriptional regulation of various important genes including tumor suppressor genes (TSGs). Recent studies indicate that the modulation of TSGs by antisense RNA may be through either up- or down- regulation, occurring either transcriptionally, post-transcriptionally or both, dependent on features of sense and antisense. Antisense RNA is the main contributor of TSG epigenetic silencing in tumorigenesis. While a general picture of the pathways involved in antisense RNA mediated gene regulation has emerged, many questions remain unaddressed: Why does some antisense RNAs function as down-regulators but others as up-regulators in different TSGs? Are the molecular mechanisms of antisense RNA regulation highly gene- and species-specific? Are they spatially and temporally restricted? Is there an antisense RNA-induced silencing complex (ARISC)? What factor(s) cause aberrant expression of antisense RNA in tumor cells? There is the potential for using antisense RNA as a biomarker for the early diagnosis of tumors and developing new therapeutic strategies for tumor treatment by targeting and controlling antisense RNA.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Stephen A Bustin, Sara Zaccara and Tania Nolan writing in Quantitative Real-time PCR in Applied Microbiology:

The real-time fluorescence-based quantitative polymerase chain reaction (qPCR) has become the benchmark technology for the detection of nucleic acids in every area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative end-point assay, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The cost of instruments and reagents is well within reach of individual laboratories, assays are easy to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to achieve accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR assay, starting with sample selection, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the significant problems associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE).

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

CFGP
CFGP (Comparative Fungal Genomics Platform) is a web-based multifunctional informatics workbench. The CFGP comprises three layers, including the basal layer, middleware and the user interface. The data warehouse in the basal layer contains standardized genome sequences of 65 fungal species. The middleware processes queries via six analysis tools, including BLAST, ClustalW, InterProScan, SignalP 3.0, PSORT II and a newly developed tool named BLASTMatrix. The BLASTMatrix permits the identification and visualization of genes homologous to a query across multiple species. The Data-driven User Interface (DUI) of the CFGP was built on a new concept of pre-collecting data and post-executing analysis instead of the 'fill-in-the-form-and-press-SUBMIT' user interfaces utilized by most bioinformatics sites. A tool termed Favorite, which supports the management of encapsulated sequence data and provides a personalized data repository to users, is another novel feature in the DUI.

MicroScope
MicroScope is a microbial genomes annotation and comparative analysis platform, which was developed by the French National Sequencing Center located at Genoscope. It is made of three major components : (i) a set of syntactic and functional annotation tools, (ii) a relational database, the Prokaryotic Genome DataBase, (PkGDB) which is linked to metabolic pathway databases (MicroCyc) created using the Pathway Tools software, and (iii) a graphical interface, the Magnifying Genome (MaGe), which allows performing relevant expert annotation that combine synteny results with metabolic network predictions.

from Kevin V. Morris writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

A growing body of evidence is beginning to emerge in human cells suggesting that particular species of non-coding RNAs can regulate gene transcription by modulating loci specific epigenetic states. While such observations were in the past relegated to imprinted genes, it is now becoming apparent that several genes in differentiated cells may be under some form of non-coding RNA based transcriptional and epigenetic control. Importantly, this form of regulation may be highly influenced by selective pressures and function in the governance and adaptability of the cell. Many studies have been carried out to date, which have begun to discern the mechanism of action whereby non-coding RNAs modulate gene transcription. Some evidence points to a role of long non-coding RNAs in controlling gene transcription by actively recruiting epigenetic silencing complexes to homology containing loci in the genome. While other studies point to a role for long intergenic non-coding RNAs in scaffold like features that are most likely equally implicit in the regulation of gene expression. The results of these studies will be considered in detail as well as the implications that a vast array of non-coding RNA based regulatory networks may be operative in human cells. Knowledge of this emerging RNA based epigenetic regulatory network has implications in cellular evolution as well as an entirely new area of pharmacopeia, namely RNA mediated epigenetic regulation of gene expression.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

In addition to QDs, luminescent dye-doped silica nanoparticles (FloDots), which consist of luminescent organic or inorganic dye molecules dispersed inside a silica matrix, have also been developed for ultra-sensitive bioanalysis and diagnostics. Owing to the silica matrix shielding effect, the doped dye molecules are protected from environmental oxygen, enabling the fluorescence to be constant and thus providing an accurate measurement for bioanalysis. Moreover, the silica matrix provides a versatile substrate for surface immobilization and various biorecognition molecules, including oligonucleotides and antibodies, have been conjugated to FloDots.

FloDots have been used successfully for the detection of DNA hybridization and in immunoassays. Using FloDots functionalized with oligonucleotides as labels for chip-based sandwich DNA assays, Zhao et al. reported a detection limit of 1 fM target DNA. The high sensitivity of the assay can be ascribed to the fact that each FloDot has the fluorescence intensity of thousands of dye molecules; therefore, each gene hybridization is reported by thousands of fluorophores. FloDot-immunoassays, using FloDots conjugated with monoclonal antibodies specific for the O-antigen of E. coli O157:H7, have also been developed to achieve rapid E. coli O157:H7 detection at the single-cell level. The fluorescence intensity emitted by one E. coli O157:H7 cell was sufficient to be detected using a normal spectrofluorometer in a conventional plate-based immunological assay, or to be accurately enumerated using a flow cytometer within 1 min of sample preparation. Although the use of FloDots represents an improvement over conventional fluorophore-based assays, they remain somewhat limited by the fundamental drawbacks of conventional fluorophores, including broad adsorption and emission profiles, which ultimately limit multiplexing capabilities.

Flow cytometry (FC) detects and quantify light scattering from fluorescent-labeled cells that have crossed a laser beam. A single sample can be analysed within 3-5 min with a quantification limit of approximately 200 cells/ml. FC, although an optical detection method, is used in combination with molecular techniques. Bacterial cells in water have been monitored with flow cytometry through nucleic acid staining or targeting specific cells with antibodies or FISH hybridization.

FC is a valuable tool to differentiate between viable, intermediate and nonviable cells. Baclightbacterial viability kit (Live/Dead kit), widely used in flow cytometry, double stains nucleic acid with SYTO dyes (green fluorescence) and propidium iodide (PI) (red fluorescence). SYTO dyes stain the nucleic acid of all the cells, resulting in green fluorescence. The cells are afterwards stained with PI which can only move into membrane compromised cells, staining the nucleic acid and resulting in red fluorescence. The disadvantage is that cells can be dead without showing membrane damage and hence is this rather an assay representing membrane damage than cell viability. Calculating the nucleic acid content has also been used as an indicator of cell viability. The theory is that cells with higher cell viability reproduces at a higher rate and therefore will contain more copies of their genome. Care must be taken with the interpretation of results obtained from this approach. Bouvier et al. investigated the varied correlation between different nucleic acid contents and metabolic activities of subpopulations from a wide range of environmental communities.

FC combined with nucleic acid staining enable researchers to investigate the growth potential of microbial pathogens in natural waters. Vibrio cholerae, the causative agent of cholera, was shown through FC and SYBR Green nucleic acid staining to grow in different freshwater samples. This contradicted previous opinions that natural waters do not have sufficient nutrients to support the growth of this pathogen. Combining these experiments with assimilable organic carbon (AOC) concentrations it was concluded that V. cholerae would proliferate in water with a minimum AOC of 60 mg/l. The same research group investigated the growth potential of E. coli O157 in freshwater samples using the same methodology. E. coli O157 was able to grow in freshwater samples with low carbon concentrations, once again contradicting previous opinions.

Fluorescence activated cell sorting (FACS) makes FC even more indispensible for detecting and differentiating between microbial pathogens in water. Cells with specific nucleic acid targets can be labeled with FISH probes, quantified and separated with FC-FACS. These cells can then be subjected for further genetic and biochemical analysis. Catalyzed fluorescent reporter disposition-FISH and molecular beacons are now incorporated into FC-FACS to increase stain sensitivity and overcome the problem of sorting cells present in low numbers. FC-FACS-FISH has also been applied to sequence previously unsequenced microorganisms and cultured previously uncultured microorganisms from environmental samples.

Real-time PCR requires monitoring the reaction during amplification. Fluorescence is a convenient method of interrogation that only requires a clear optical path for excitation and emission. Double-stranded DNA (dsDNA) dyes and fluorescently-labeled probes are both commonly used. dsDNA dyes directly measure the amount of double-stranded product produced. Probes used in real-time PCR function indirectly through fluorescence resonance energy transfer (FRET) or fluorescence quenching. Initially proposed in the late 1940s, it was not until the 1980s that FRET was applied to DNA (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). However, real-time monitoring with probes was only achieved several years later after dsDNA dyes were established in real-time PCR. One advantage of probes over dsDNA dyes is multiplexing by color with different fluorescent dyes. Nevertheless, this advantage comes at a cost in instrumentation and analysis complexity. Furthermore, multiplex analysis with dsDNA dyes is possible by melting temperature separation of products and/or probes.

from Andre Irsigler and Karen M. McGinnis writing in Epigenetics: A Reference Manual:

Maize has served as an excellent model for the study of epigenetic gene regulation for the past several decades. The pioneering work of maize geneticists like Barbara McClintock, Alexander Brink, Marcus Rhoades, and others led to the observation of many fascinating phenomena that were later demonstrated to be epigenetically regulated events. Since these observations were made, a great deal of progress has been made in determining the underlying causes of the phenomena, and many of these examples of epigenetic gene regulation are currently being used to elucidate the mechanisms of epigenetic heritability in plants. Today, these phenomena and the mutants that impact them serve as resources for studying how DNA methylation, chromatin structure, and small RNAs act to influence paramutation, gene silencing, and parent-of-origin dependent dosage compensation. The key attributes and potential contributions of each resource are discussed in the context of understanding the mechanisms and significance of epigenetic gene regulation in large, complex genomes.

Further reading: Epigenetics: A Reference Manual

Comparative analysis is an increasingly important step in the annotation and analysis process of genome sequence data, allowing phenotypic differences between strains and species to be correlated with changes in the chromosomes. For example, comparative sequence analysis has enabled the identification of cis-regulatory regions and location of coding exons using purely computational means. Visual front-ends are necessary and important to make the process of viewing alignments intuitive and easy to facilitate discovery of conserved sequences for functionally significant regions. Below we describe a few visualization tools for genome comparisons.

PipMaker and MultiPipMaker
PipMaker is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. The web site also provides a plot of the locations of those segments in both species. PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. PipMaker supports analysis of unfinished or working draft sequences by permitting one of the two sequences to be in un-oriented and unordered contigs. Similarly, MultiPipMaker allows the user to visualize relationships among more than two sequences. All pairwise alignments with the first sequence are computed and then returned as interleaved pips. Moreover, MultiPipMaker can be requested to compute a true multiple alignment of the input sequences and return a nucleotide-level view of the results.

ACT
ACT (Artemis Comparison Tool) is a DNA sequence comparison viewer, such as parsed BLAST alignments based on Artemis - an annotation tool. Similar to other Artemis tools, ACT is written in Java and runs on Unix, GNU/Linux, Macintosh and MS Windows systems. It can read complete EMBL and GENBANK entries or sequence in FASTA or raw sequence format. Other types of readable sequence input files include EMBL, GENBANK and GFF formats. The sequence comparison displayed by ACT is usually the result of running a blastn or tblastx search.

VISTA
Vista (Visualization and Alignment Software for Comparative Genomics) is a visualization tool for alignments, which displays GLASS alignments. It is a program to depict long alignments of DNA sequences from two or more organisms with various types of annotation in a clear and easily interpretable format. Originally it was developed to locate conserved sequences in syntenic regions of different genomes. The key features of the VISTA program are mainly the following:
1. Clean graphical output, allowing for easy identification of sequence similarities and differences.
2. Easily configurable, enabling the visualization of alignments of up to several million bases at different levels of resolution.
3. Displays alignments of draft sequences.
4. Displays sequence annotations such as repeats, coding exons, UTRs and more.
The VISTA plot is based on moving a user-specified window over the entire alignment and calculating the percent identity over the window at each base pair.

SynPlot
Synplot (displays DIALIGN and GLASS alignments) is an application program, written in Perl, for viewing global alignments of syntenic regions of genomic DNA sequence. The alignment is used to calculate the percentage identity along the alignment within a sliding window, the width of which can be specified by the user. This information is used to draw a picture of the alignment in postscript format. The sequences are rendered as lines interrupted by spaces corresponding to the gaps introduced by the alignment, with a plot of the percentage identity underneath. Features can also be drawn on the sequence lines. This program uses a GFF format file output by ACeDB from the annotated genomic sequence, and a configuration file which specifies the color, height and order in which the rectangles representing the features are drawn.

from Chihiro Kohama and Hidenori Kiyosawa writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

Genomic DNA and full-length cDNA sequencing projects have provided fundamental data that have led us to the novel notion that antisense transcription is a universal phenomenon in many organisms, including mammals and plants. Successive expression analyses utilizing microarrays, genome-tiling arrays, and recent RNA sequencing with next-generation sequencers have supported this concept. We review how these genome-wide, natural antisense transcript analyses have proceeded, and we then introduce representative examples of the known functions and functional implications of antisense transcripts. The variety of modes in which antisense transcripts function in cells reveals that these transcripts do not constitute a uniform group, but perform various types of gene expression regulation.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

Genome sequence comparison has been an important method for understanding gene function and genome evolution since the early days of gene sequencing. Alignment of DNA sequences is the core process in comparative genomics. In recent years, an important new sequence-analysis task has emerged: comparing an entire genome with another. Several powerful alignment algorithms have been developed to align two or more sequences.

MUMmer
MUMmer is a system for rapidly aligning entire genomes, whether in complete or draft form. MUMmer can also align incomplete genomes; it can handle thousands of contigs from a shotgun sequencing project, and will align them to another set of contigs or a genome using the NUCmer program included within the system. If the species are too divergent for a DNA sequence alignment to detect similarity, then the PROmer program within the environment can generate alignments based upon the six-frame translations of both input sequences. The original MUMmer system, version 1.0, was described in a 1999 Nucleic Acids Research paper. Version 2.1 appeared a few years later and was described in a 2002 Nucleic Acids Research paper, and the most recent version MUMmer 3.0 was described in a 2004 Genome Biology paper.

BLAT
BLAT (The BLAST-Like Alignment Tool) is a new tool for sequence alignment, which is similar in many ways to BLAST. The program rapidly scans for relatively short matches (hits), and extends these into high-scoring pairs (HSPs). However, BLAT differs from BLAST in several significant ways. Specifically, where BLAST builds an index of the query sequence and then scans linearly through the database, BLAT builds an index of the database and then scans linearly through the query sequence. Where BLAST triggers an extension when one or two hits occur in proximity to each other, BLAT can trigger extensions on any number of perfect or near-perfect hits. Where BLAST returns each area of homology between two sequences as separate alignments, BLAT stitches them together into a larger alignment. Both the client/server and the stand-alone can do comparisons at the nucleotide, protein, or translated nucleotide level.

MEGABlast
Mega BLAST uses the greedy algorithm of Zhang et al. for nucleotide sequence alignment search and concatenates many queries to save time spent scanning the database. This program is optimized for aligning sequences that differ slightly as a result of sequencing or other similar "errors". It is up to 10 times faster than more common sequence similarity search and alignment programs and therefore can be used to swiftly compare two large sets of sequences against each other.

from Marcel W. Coolen and Susan J. Clark writing in Epigenetics: A Reference Manual:

Over the past decades it has become ever more apparent that understanding the genome did not stop with unravelling the genetic code. Regulatory mechanisms are needed to determine which parts of the genome are active or inactive, and to form a memory system that can be passed on over multiple cell divisions for proper functioning of the cell. These mechanisms underpinning heritable gene regulation are encapsulated by the term "epigenetics" and include histone modifications, miRNA and non-coding RNA expression, and methylation of cytosine residues in DNA. In this review, we compare the various methods that can be used to analyse DNA methylation patterns throughout the genome, and discuss their advantages and disadvantages. In addition, we present a detailed protocol for genome-wide DNA methylation analysis based on the capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein combined with second generation sequencing.

Further reading: Epigenetics: A Reference Manual

January 17 - 22, 2012 Epigenomics
Keystone Symposium. This meeting will highlight recent advances in the application of genomics techniques to the study of epigenetics.

January 20 - 22, 2012 RNA-UK 2012
RNA-UK is a biannual meeting that focuses on the increasingly important functions of RNA. The meeting will focus on current research on RNA processing, RNA-mediated gene regulation, RNA localisation, RNA stability/decay, RNA function and RNA structure.

February 7 - 12, 2012 Gene Silencing by Small RNAs
Keystone Symposium. The impact of small non-coding RNAs has profoundly touched the fields of development and cell biology, functional genomics, human disease and drug therapy. This mode of gene regulation is not restricted to eukaryotes; bacteria utilize small RNAs, notably those made from CRISPR loci that silence the expression of bacteriophages, transposons and plasmids.

March 25 - 27, 2012 microRNA 2012: International Symposium
Covers various cover themes on microRNA research.

March 27 - 29, 2012 RNAi 2012
The 7th Annual Oxford RNAi Conference, RNAi2012, will address most aspects of biology and application of RNA interference, and welcomes proposals on endogenous and exogenous roles of small RNAs in gene regulation, health and disease.

March 31 - April 5, 2012 Non-Coding RNAs
Keystone Symposium. The goal of the Keystone Symposia meeting on Non-Coding RNAs is to highlight the similarities and differences in various model systems to better understand the role of long ncRNAs in the regulation of genomes.

April 21 - 27, 2012 Analysis of small non-coding RNAs
EMBO Practical Course. Analysis of small non-coding RNAs: From massively parallel sequencing to in-situ hybridization, from discovery to validation.

May 1 - 2, 2012 RNAi, MicroRNAs 2012
International conference at the cutting edge of the life-science industry.

June 11 - 16, 2012 Antiviral RNAi: From Molecular Biology Towards Applications
ESF-EMBO Symposium. RNAi-based strategies have been used to engineer plants that resist a large variety of viruses, and administration of antiviral small RNAs has been demonstrated to be highly efficient in inhibiting viral infections in mammals, and a number of clinical trials are currently ongoing to assess the safety and efficacy of this therapeutic strategy.

July 9 - 10, 2012 Epigenomics 2012
International conference at the cutting edge of the life-science industry.

September 16 - 19, 2012 The reciprocal interactions of signalling pathways and non-coding RNA
EMBO Workshop.

Full details at our conference website Non-coding RNA and Epigenetics Conferences

from Robert Pon writing in Bacterial Glycomics: Current Research, Technology and Applications:

Abstract to follow

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

Metal nanoparticles, such as gold, silver and iron, constitute one of the most researched branches of nanotechnology due to their electronic, optical, catalytic and thermal properties. Among these, gold nanoparticles are intensively used in a variety of colorimetric and fluorescence biodetection assays. Specific focus has been directed at colloidal gold nanoparticles ranging from 3 to 100 nm in size, since they are rather stable and their properties can be easily tailored by chemical modification of their surfaces.

Gold nanoparticle-based probes have been used in the identification of pathogenic bacteria in a chip-based system. The assay consists of a capture DNA strand immobilized on a glass chip that recognizes the DNA of interest. A separate sequence on the captured target strand is then labelled with an oligonucleotide-functionalized gold nanoparticle probe. After catalytic reduction of silver onto the gold nanoparticle surfaces to amplify the target signal, the capture-strand/target/nanoparticle sandwich is visualized with a flatbed scanner. The target DNA was detected at concentrations of 50 fM, which represents a nearly 100-fold increase in sensitivity over traditional fluorescence-based assays. Gold nanoparticles have also been used in the detection of genomic DNA from Staphylococcus aureus, without enzymatic amplification, at concentrations of 33 fM. The assay depends upon the target-selective binding of gold nanoparticle probes to consecutive regions of a target DNA sequence in solution, followed by transfer to an evanescently illuminated glass microscope slide for light scattering measurements. Whereas the gold particle probes scatter orange light in the presence of target, the probes scatter green light if the target is absent, whilst the target concentration is quantified by measuring the intensity of scattered light. Multiplexed detection of different viruses and bacteria, using gold nanoparticles surface functionalized with a target-specific oligonucleotide and further encoded with a Raman-active dye molecule, has also been demonstrated. Whereas the complementary probe sequence imparts the specificity for the target sequence of interest, the presence of the target is confirmed by silver staining and the identity of the target is revealed by detecting the surface-enhanced Raman scattering (SERS) of the Raman dye near the nanoparticle surface. This assay demonstrated a sensitivity of 1 fM target concentration.

Despite its widespread use in nucleic acid hybridization assays, the use of gold nanoparticles for detection of bacterial pathogens in immunoassays has only recently been described. The assay comprises detection of organism-specific antigens with biotinylated polyclonal antibodies after which gold particles functionalized with a secondary antibiotin antibody are added, and the particles are then visualized under a dark-field stereomicroscope. The immunoassays were demonstrated to reliably detect Helicobacter pylori and E. coli O157:H7 antigens in quantities in the order of 10 ng, which provides a sensitivity of detection comparable to those of conventional dot blot assays. A two dot filter system has also been developed where colloidal gold nanoparticles (2 nm) were bounded onto anti-E.coli O157:H7 antibodies. These monoclonal antibodies were bounded onto a 0.2 microm nitrocellulose filtermembrane through which water was filtered. The same antibodies that captured the bacteria acted as detectors since the gold nanoparticles could be visualized under epifluorescence. This one-step detection method not only had a low detection rate (1 cfu/100 ml) and high specificity but would be inexpensive and easy implemented in the routine testing of water samples.

from Rakesh Kumar Singh, Johanna Paik and Akash Gunjan writing in Epigenetics: A Reference Manual:

In eukaryotes, the genetic material in the form of DNA is wrapped around histone proteins to form nucleoprotein filaments called chromatin. Histones help package the DNA to fit it inside the nucleus of each cell, which in turn regulates access to the genetic information contained within the DNA. Hence, all DNA transactions are likely to be affected by histone metabolism. Eukaryotes carry multiple histones genes that can potentially generate enormous quantities of histone proteins. When present in excess, the positively charged histones can potentially "stick" non-specifically to the negatively charged DNA and adversely affect processes that require access to DNA. Not surprisingly, aberrant histone stoichiometry, chromatin assembly or chromatin structure lead to genomic instability, which is characterized by the increased rate of acquisition of alterations in the genome and is associated with deleterious human conditions such as cancer and aging. Hence, histone synthesis is coupled to ongoing DNA replication and is regulated transcriptionally and posttranscriptionally. To further avoid the deleterious consequences of excess histones, a posttranslational regulatory mechanism was described recently whereby excess histones are targeted for degradation by the ubiquitin-proteasome system. In this chapter, we discuss the causes and consequences of excess histone accumulation, as well as the strategies that cells have evolved to deal with them.

Further reading: Epigenetics: A Reference Manual

from Olga M. Pena, John D. F. Hale and Robert E.W. Hancock writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

The increasing problem of resistance to antimicrobial agents, combined with the limited development of novel agents to treat infectious diseases is a serious threat to human morbidity and mortality around the world. Among the available strategies available to create new therapeutic agents is the enhancement of the multifunctional properties of the natural anti-infectives, cationic host defense (antimicrobial) peptides (HDPs). This chapter will provide a summary of our current understanding of the different types of HDPs including natural and synthetic peptides and their antimicrobial and immunomodulatory modes of action. Additionally, we will describe new approaches to peptide design and discuss both the therapeutic potential and prospective challenges in the utilization of peptides for antibacterial

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

In contrast to hydrolysis probes, the fluorescence from hybridization probes is reversible and depends only on probe hybridization. The first hybridization probes used in real-time PCR were dual hybridization probes consisting of two oligonucleotides, one labeled at the 3'-end the other at the 5'-end. Upon hybridization to their complementary sequences and fluorescent excitation, FRET increases. Signal generation with dual hybridization probes requires annealing of four oligonucleotides (two primers and two probes), suggesting even better specificity than hydrolysis probes. Later, single hybridization probe designs were developed, including FRET between an internally labeled primer and a single-labeled probe and deoxyguanosine quenching of a single-labeled probe (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). In contrast to hydrolysis probes that are consumed during amplification, the fluorescence of hybridization probes is reversible, enabling melting analysis. The first FDA-approved genetic tests in the US (F5 and F2 single base variants) used dual hybridization probes and melting analysis for genotyping.

In 1991, Holland and colleagues at the Cetus Corporation used the 5' to 3' exonuclease activity of Taq polymerase to detect amplification products post-PCR. An oligonucleotide probe complementary to the PCR product was used with a non-extendable 3'-end and a radioactively labeled 5'-end. During amplification the polymerase degraded the probe, releasing the radioactive label as smaller fragments of the probe. However, a post-PCR radiograph was required in order to visualize the degraded probe. By replacing the radioactive label with two fluorescent labels in a FRET relationship, successful allele discrimination and later real-time monitoring were achieved. These dual-labeled fluorescent probes were hydrolyzed by the 5' to 3' exonuclease activity of Taq during PCR, separating the fluorescent labels with a loss of FRET to generate fluorescence. Specificity was enhanced over dsDNA dyes because complementation to three independent oligonucleotides (two primers and one probe) was necessary for probe hydrolysis and signal generation. Hydrolysis probes (also known by the trademark TaqMan, among others) are the most commonly used probes today (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Their popularity was advanced by simplified design and a strong commercial effort to provide synthesis services. Signal generation is produced by probe hydrolysis and is irreversible and cumulative.

from Nichollas E. Scott, Stuart J. Cordwell, John F. Kelly and Susan M. Twine writing in Bacterial Glycomics: Current Research, Technology and Applications:

There are increasing numbers of reports of bacterial glycosylation in pathogenic bacteria, with well-characterized bacterial glycoproteins including pilins, flagellin and other surface-associated proteins. However, the discovery of bacterial glycoproteins can be challenging due to the diversity of glycans bacteria use to modify proteins. At the protein level, so-called 'top-down' mass spectrometry studies of intact protein can rapidly characterize bacterial glycan ions. At the peptide level, interpretation of individual bacterial glycopeptide tandem mass spectra can be challenging, owing to the diverse range of bacterial glycans produced. Reports of methods to specifically isolate bacterial glycopeptides are advancing knowledge of bacterial glycoproteomes. Herein, we provide an overview of protein and peptide centric mass spectrometry and related analytical techniques for the enrichment and analysis of bacterial glycoproteins.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

Immunological methods are based on the specific recognition between antibodies and antigens, and the high affinity that is characteristic of this recognition reaction. Consequently, many different immunoassay methods have become available for both quantitative and qualitative analysis of pathogenic bacteria in water. These include immunocapture of cells or antigens by enzyme-linked immunosorbent assay (ELISA or EIA), or detection of targeted cells by immunofluorescence (IFA). These assays can be performed by a direct or indirect manner. In a direct immunoassay, the monoclonal or polyclonal antibodies, directed against antigens located on the surface of the target pathogen (such as capsid proteins, cell wall or flagellar antigens), are conjugated with a fluorochrome or fluorescent dye. Alternatively, secondary enzymatically- or fluorescently-labelled antibodies directed against the primary antibodies (now serving as antigens) can be used in an indirect immunoassay. The advantage of this procedure is that the secondary antibodies can easily be obtained from a commercial supplier with a range of conjugated fluorochromes and it leads to signal amplification as several labelled secondary antibodies can bind to a single unlabelled primary antibody. The antigen-antibody complex is detected and quantified by the ability of the enzyme to react with a substrate that produces either a coloured product for colorimetry or emits light for luminometry. The immunoassays are often performed on a solid phase to which the pathogen antigens have been applied, such as a membrane filter or the bottom of a microtitre plate well.

Studies have shown that solid-phase enzyme immunoassays generally are too insensitive for direct detection of microbial pathogens in water, as they require a minimum of 103 to 104 target microbes (or their antigens) for detection. In most situations drinking water and its sources rarely contain high enough levels of most target pathogens for direct immunoenzymatic detection. Nevertheless, enumeration of diluted specific cells can be obtained by means of immunomagnetic separation (IMS). Immunomagnetic separation, also termed immunocapture or antibody capture, is a method that uses paramagnetic synthetic beads or other magnetic particles that have been coated with monoclonal or polyclonal antibodies directed against the target microbes to recover the microbes from the sample by antigen-antibody reactions. The retained microbes can be analyzed directly or after they or their nucleic acids have been released or extracted from the antibody and solid phase by various physical or chemical methods. IMS methods have the advantage of selecting, separating and purifying specific target microbes from other microbes and from solutes, based on the specificity of the antigen-antibody reaction. This is a powerful approach for recovering, enriching, purifying and concentrating the target viruses, bacteria and parasites from the sample matrix. However, it is not applicable to some pathogens because of the lack of antisera or the antigenic diversity of a large pathogen group lacking a common antigen and thus requiring many antisera.

As an alternative to the above assays, agglutination methods can be used to detect pathogens by combining dispersed microorganisms with antibodies (on a slide, for example) and allowing for antigen-antibody reactions to produce agglutination (clumping) that can be scored as negative or various degrees of positive. One modification is latex bead agglutination in which antibodies against a specific microbial antigen are attached to latex beads. The beads are reacted with the sample and should the sample contain the specific antigen, agglutination occurs by the reaction of antigens with antibodies on the beads resulting in the beads clumping together. As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples. The target microbes must first be cultured in order to obtain a sufficient number of them or a sufficient amount of antigen to detect and antigenically characterize them by agglutination methods.

The use of immunological methods for the detection of specific microorganisms is a rapid and simple technique, the accuracy of which mainly depends on the specificity of the antibody. Nevertheless, its application to the detection of specific microorganisms from environmental water samples is limited. While IFA allows specific identification and detection at a single-cell level, it does not provide information on the physiological status or viability of the detected cells. The ELISA is a rapid, simple and quite sensitive test. However, assay limitations are often associated with the specificity of the antibody used, the concentration of both antibody and antigen, and the solid matrix often leads to non-specific binding of the antigen or of the secondary antibody.

Monoclonal antibodies are better suited for biosensors because of their higher specificity. Polyclonal antibodies recognize different epitopes on the same pathogen. False positives can be generated when these antigens are present in other closely-related non-pathogenic microorganisms. The thermal instability of antibodies, in particular monoclonal antibodies, is another drawback when applying them in environmental biosensors. Single domain antibodies (also referred to as nanobodies) have however been developed that are thermostable, even at temperatures as high as 90 degrees C. Their small size, high solubility and refolding capacity are other features that make them ideally situated for biosensing applications.

from Stéphane Labialle, Patrice Vitali, and Jérôme Cavaillé writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

Genomic imprinting is a developmentally controlled form of epigenetic regulation that triggers parent-of-origin specific expression of a few mammalian genes. That is, for a given gene, only one of the two parental alleles is transcriptionnally active. Over the last several years, many imprinted small regulatory non-coding RNAs (ncRNAs, including microRNAs and box C/D small nucleolar RNAs) have been described at four evolutionarily distinct imprinted loci: the Snurf-Snrpn/Prader-Willi Syndrome and Dlk1-Dio3 chromosomal domains, and more recently at the C19MC locus and the Sfmbt2 gene. Remarkably, many imprinted ncRNA loci are clustered within large arrays formed by tandemly-repeated genes of related sequences and processed from long non-coding transcripts extending over hundreds of kilobases. Imprinted gene loci therefore give rise to unique opportunities to address both the impact of small and long non-coding RNA genes on the evolution, expression and function of mammalian genomes. In this chapter, we survey our current understanding of the functions of imprinted ncRNAs, with a particular attention to their suspected involvement in the Prader-Willi disease and higher-brain functions, as well as to their hypothetical contribution in the evolution and/or control of genomic imprinting.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Sarah A. Kinkel and Hamish S. Scott writing in Epigenetics: A Reference Manual:

DNMT3L (DNA methyltransferase 3 like) is member of the DNA methyltransferase family of enzymes responsible for the methylation of CpG dinucleotides. Biochemical studies have revealed that while DNMT3L lacks DNA methyltransferase activity, it can bind to and stimulate the activity of de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3L has also been shown to interact directly with chromatin via its plant homeodomain (PHD)-like zinc finger domain. Studies in Dnmt3L-deficient mice have revealed that DNMT3L is essential for establishing correct methylation patterns at RetroTransposable Elements (RTE), unique loci and parentally imprinted genes in germ cells, and mice without DNMT3L are rendered infertile. Female Dnmt3L^-/- mice have apparently normal meiosis but in male Dnmt3L-/- germ cells there is asynapsis of chromosomes, and "meiotic catastrophe". Dnmt3L was among the first mammalian genes shown to have a paternal effect, where the genotype of the father (Dnmt3L^+/-) affects sex chromosome aneuploidy in adult and embryonic offspring. This chapter will discuss the role of DNMT3L in establishing DNA methylation patterns during gametogenesis, as well as the proven and potential consequences of DNMT3L-deficiency to fertility and somatic and germline genetic disease in light of the increasing evidence that epigenetic reprogramming is a dose sensitive and partially stochastic process.

Further reading: Epigenetics: A Reference Manual

Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection Edited by: Kevin V. Morris ISBN: 978-1-904455-94-3 Publisher: Caister Academic Press Publication Date: February 2012 Cover: hardback

read more ...]]>http://www.caister.com/molecular-biology-blog/2011/10/new-epigenetics-book-available-soon.htmlhttp://www.caister.com/molecular-biology-blog/2011/10/new-epigenetics-book-available-soon.htmlWed, 05 Oct 2011 12:55:47 GMTLipopolysaccharide Biosynthesis

from Leslie Cuthbertson writing in Bacterial Glycomics: Current Research, Technology and Applications:

Lipopolysaccharide (LPS) constitutes the major portion of the outer leaflet of the outer membrane and plays a major role in the physiology of Gram-negative bacteria. LPS can be divided into three structurally distinct regions: lipid A, core oligosaccharide and O-antigenic polysaccharide. Each of these regions as well as regulated modifications, are important in the overall functions of the LPS molecule. Synthesis of lipid A and the core oligosaccharide occurs in the cytoplasm and is separate from that of the O-antigenic polysaccharide. These two portions of the LPS molecule are then ligated in the periplasm prior to transport to the outer membrane. This chapter will describe the structure and cytoplasmic synthesis of LPS, modifications to these structures regulated by environmental conditions or phage-encoded genes, and the transfer of LPS to its final destination at the cell surface.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

December 5 - 5, 2011 The Use of Zinc Finger Nucleases for the Development of Next Generation Cell Lines and Animal Models
Heidelberg, Germany Further information
Featuring keynote presentations, this symposium will allow participants to discover the latest applications of transgenics and cell based models for the study of gene regulation and disease.

December 13 - 16, 2011 The 34th Annual Meeting of the Molecular Biology Society of Japan MBSJ
Pacifico Yokohama, Japan Further information
Molecular Biology Society of Japan MBSJ
Suggested reading: | Molecular Biology books

]]>http://www.caister.com/molecular-biology-blog/2011/10/molecular-biology-conferences-december-2011.htmlhttp://www.caister.com/molecular-biology-blog/2011/10/molecular-biology-conferences-december-2011.htmlTue, 04 Oct 2011 13:34:58 GMTMolecular Biology Conferences Marco Island, FL, USA Further information
RAFT IX 2011. Recent Advances in Fermentation Technology
Suggested reading: | Molecular Biology books

November 8 - 9, 2011 Advances in Metabolic Profiling
Dublin, Ireland Further information
Select Biosciences. The 7th annual Advances in Metabolic Profiling conference and exhibition.

November 8 - 9, 2011 Mass Spec Europe
Dublin, Ireland Further information
Select Biosciences. The 3rd annual Mass Spec Europe conference and exhibition.
Suggested reading: | PCR Troubleshooting and Optimization: The Essential Guide

November 14 - 18, 2011 Computational structural biology: from data to structure to function
Hinxton, UK Further information
EMBO Practical Course.
Suggested reading: | Bioinformatics books

November 15 - 18, 2011 Life Sciences And The Issues Of Our Time
Bethesda, Md, USA Further information
ASBMB Special Symposium Series. The ASBMB Special Symposia program was established to provide specific or under-represented segments of the scientific community with opportunities to present unique, cutting-edge science and engage in active networking opportunities. Meeting attendees include graduate students, postdocs, investigators and industry professionals.
Suggested reading: | Molecular Biology books

November 28 - 30, 2011 The 2nd international conference of Human Genetics and Genome Research Division
Cairo, Egypt Further information
The scientific program will deliver breakthrough advances and insight in scientific and clinical research, patient management and practice through an outstanding range of scientific and educational symposia, special sessions, teaching lectures, workshops, oxford style debates and more.
Suggested reading: | Molecular Biology books

]]>http://www.caister.com/molecular-biology-blog/2011/10/molecular-biology-conferences-november-2011.htmlhttp://www.caister.com/molecular-biology-blog/2011/10/molecular-biology-conferences-november-2011.htmlTue, 04 Oct 2011 13:34:22 GMTLong Non-coding RNAs (lncRNAs) and Cancer

from Jessica M. Silva and David I. Smith writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

There are numerous non-coding transcripts in addition to those housekeeping transcripts (ribosomal RNAs and transfer RNAs) that participate in the processes of protein production within the cells. These include the large number of small RNAs such as microRNAs, piwiRNAs and snoRNAs. In addition to these relatively small transcripts there are also considerably longer non-coding transcripts that also play important roles within the genome. These long non-coding RNAs (lncRNAs) can be differentiated from each other based upon where they are derived from within the genome. For instance, there are intronic lncRNAs (transcribed between exons of genes), intergenic lncRNAs (transcribed in the space between genes), and lncRNAs that are more complex as they overlap both intron and exon of a coding gene. Each of these lncRNAs may also be in the sense or in the antisense direction. The list of long non-coding RNAs (lncRNAs) that are associated with diseases is rapidly increasing. Some lncRNAs have been found to be aberrantly expressed in various diseases including psoriasis, mental disorders, autism, and also cancer. The list of lncRNAs associated with cancer is increasing. However, relatively little is known about the precise functions of most cancer associated lncRNAs, but of what is known suggest that these long non-coding transcripts function in many different ways within cells and promote cancer development and progression. We will review what is known about the role that some of the lncRNAs play in cancer

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Brian P. Chadwick writing in Epigenetics: A Reference Manual:

The recent completion of several mammalian genome sequences makes obvious that we share a near-identical collection of genes. What defines us as human must therefore be encoded within regions of the genome where we differ, providing an added level of complexity that probably influences the spatial and temporal expression of genes. Most DNA sequence variation occurs within the repetitive DNA, once called 'Junk DNA' that accounts for at least half of the human genome, and evidence is mounting for its important role in genome function. Although some repeat elements are conserved to some extent between mammals, their precise copy number and genomic location typically are not. In addition, some repeats are not conserved, including the large tandem repeats. This chapter focuses on two large tandem arrays in the human genome that can adopt quite different chromatin configurations as a result of epigenetic changes; one as a direct consequence of X chromosome inactivation and the other in the context of disease susceptibility. Both cases highlight how alternate packaging of these unusual DNA sequences probably results in differing functions. In each instance, common denominators are the acquisition of the epigenetic organizer protein CTCF and a distinct change in transcripts originating from the array.

Further reading: Epigenetics: A Reference Manual

In contrast to gold nanoparticles and QDs, magnetic nanoparticles have not been used in many biological applications. Nevertheless, advances in the synthesis of monodispersed magnetic nanoparticles, ranging in size from 2 to 20 nm, has provided a basis from which to explore applications of magnetic nanoparticles in diagnostics. Magnetic nanoparticles are produced from materials that can be strongly attracted by magnets or be magnetized. They can be prepared in the form of single domain or superparamagnetic (Fe₃O₄), greigite (Fe₃S₄), maghemite (gamma-Fe₂O₃), and various types of ferrites (MeO.Fe₂O₃, where Me = Ni, Co, Mg, Zn, Mn, etc.). Bound to biorecognition molecules, magnetic nanoparticles can be used to facilitate the separation, purification and concentration of different biomolecules. To do so, biorecognition molecules such as antibodies can be immobilized on the surface of magnetic nanoparticles through covalent or electrostatic interactions. After reacting these magnetic nanoparticles with sample solutions, targeted molecules can be bound by or captured on the surface of these magnetic nanoparticles. By applying a magnetic field, these nanoparticles can subsequently be concentrated and separated from the bulk solution and identified.

Biofunctional magnetic nanoparticles, in which thiolated vancomycin was attached to FePt nanoparticles, have been used to capture and detect of a wide range of bacteria at very low concentrations within 60 min. These included capturing and detection of Staphylococcus aureus at 8 cfu/ml, S. epidermidis at 10 cfu/ml, Enterococcus faecalis at 26 cfu/ml, and E. coli at 15 cfu/ml. Although the sensitivity achieved using magnetic nanoparticles is comparable to PCR-based assays, the direct capture protocol is faster than PCR when the bacterium count is low since it obviates the need for pre-enrichment of bacteria through culturing. In an alternative approach, Ho et al. reported combining biofunctional magnetic nanoparticles with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect pathogenic bacteria in water. Biofunctional nanoparticles were fabricated by attaching human immunoglobulin (IgG), which binds selectively to IgG-binding sites on the cell walls of pathogens, onto the surfaces of magnetite (Fe₃O₄) nanoparticles. Using this assay, both S. saprophyticus and S. aureus were detected at concentrations of 3ˆó10⁵ cfu/ml in aqueous sample solutions. Measuring adenosine triphosphate (ATP) bioluminescence of bacteria captured onto magnetic nanoparticles is another proposed method for detecting microorganisms. E coli was detected in milk by Cheng et al. within a short period (1 h) and with a low detection limit (20 cfu/ml).

Biofunctional magnetic glyconanoparticles have also been engineered by covently binding unmodified monosaccharide d-mannose onto iron oxide nanoparticles. These particles had the ability to recognize mannose-specific receptor sites on E. coli. Magnetic nanoparticles have been developed to sequester DNA in water and capture the DNA-nanoparticles complexes by the application of high-gradient magnetic separation. Modifying magnetite clusters with poly(hexamethylene biguanide)- and polyethyleneimine resulted in strong cationic nanoparticles which enabled the binding with DNA molecules through electrostatic forces. The cationic nanoparticles can also serve as a disinfectant by binding to the negatively charged cell envelopes of bacteria. These particles were colloidally stable in fresh and ocean water for weeks at a pH <= 10.

Magnetic microparticle-antibody conjugates (Dynabeads) are commercially available and kits have been developed for the detection of Legionella species, Cryptosporidium oocysts and Giardia cysts from concentrated water samples. Dynabeads are also available for the detection of E. coli, Salmonella and Listeria species; however the samples must be grown for 6 - 8 h in a pre-enrichment broth. Streptavidin coated Dynabeads allow researchers to design their own magnetic microparticle-antibody conjugates for specialized assays (www.invitrogen.com). Biotinylated organism-specific antibodies will bind covalently onto the streptavidin coated Dynabeads. A wide range of biotin-labeled antibodies are available from companies such as Abcam (www.abcam.com).

Despite the promise shown by biofunctional magnetic nanoparticles, some challenges regarding their widespread use have yet to be overcome. In addition to requiring a robust surface chemistry to attach bioactive molecules onto magnetic nanoparticles without laborious synthetic efforts, more precise control of the numbers and orientations of the molecules on the surfaces of magnetic nanoparticles is also required.

Melting curve analysis is a powerful and practical extension of real-time PCR. While real-time PCR focuses on collecting fluorescence at a single temperature each PCR cycle, melting analysis monitors fluorescence over time as the temperature is changing. Melting analysis fits nicely into the kinetic paradigm of PCR. Duplexes melt as the temperature increases, and the hybridization of both PCR products and probes can be monitored. Similar to "old" (slow) PCR being considered an equilibrium process, "old" (dot blot) hybridizations were performed at a single temperature. Dynamic monitoring of the entire melting curve as the temperature changes defines the entire melting transition, not just a single point (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Melting curve analysis was first integrated with real-time PCR on the LightCycler. No separations or reagent additions were required and melting analysis was fast (typically 2-15 min). The dye SYBR Green I conveniently provided quantification during PCR and melting analysis after PCR. The melting temperature of a DNA duplex is determined in large part by its sequence, G/C content and length. Specific PCR products can be easily distinguished from nonspecific PCR products. In many cases melting analysis eliminates the need for post-PCR processing such as gel electrophoresis. Genotyping by melting analysis was first demonstrated with a single hybridization probe and FRET to monitor probe melting. Different single base variants produced different probe stabilities, which were revealed by melting analysis. Later, dual hybridization probes were used for genotyping and both color and temperature multiplexing exploited. The use of a single fluorescein-labeled probe instead of two probes was a further simplification. Genotyping by melting without labeled probes was first shown with allele-specific PCR and SYBR Green I. Three primers were used, one with a GC-tail to discriminate alleles by melting temperature. Genotyping without GC-tails or labeled probes became possible with the availability of saturation dyes that detect heteroduplexes. These methods are detailed later in the section on high resolution melting analysis.

from Danielle H. Dube writing in Bacterial Glycomics: Current Research, Technology and Applications:

Though long believed to be absent from bacteria, glycoproteins are now known to be synthesized in a number of bacterial species. Traditional methods to study glycoproteins have revealed fascinating glycan structures that are exclusively found in bacteria and are frequently linked to pathogenesis. In recent years, these methods have been augmented by a complementary approach, termed metabolic oligosaccharide engineering (MOE), to facilitate large scale systematic studies of the entire complement of glycan structures in bacteria, referred to as bacterial glycomics. In MOE, bacterial glycans are metabolically labeled with unique chemical functionalities, called chemical reporters. Labeling bacterial glycans in this manner facilitates glycoprotein detection and enrichment. In addition to enabling glycoprotein profiling, the labeled glycans can undergo selective covalent bond formation, thereby permitting further applications. For example, labeled glycans are poised to disrupt the bacterial surface coat, target bacterial cells with toxins, trap glycan-based host-pathogen interactions, and image dynamic changes in glycosylation. This chapter focuses on MOE methodology, its application to the study of bacterial glycoproteins, and its future role in treating infectious disease.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Elaine R. Lee, Kenneth F. Blount and Ronald R. Breaker writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

The need for new antibacterial drug targets increases as antibiotic resistant pathogens continue to arise. Researchers have recently begun to investigate whether structured noncoding RNAs such as riboswitches can be exploited as targets for new classes of antimicrobial compounds. Riboswitches are gene control elements made entirely of RNA, and in bacteria they are usually located in the 5' untranslated regions (UTRs) of messenger RNAs. These elements are capable of forming complex structures that selectively bind to specific fundamental metabolites and often control the expression of proteins critical for bacterial metabolism and survival. In principle, novel ligands could be designed that target specific riboswitches and alter the expression of the critical genes they regulate. Several riboswitch classes have begun to be examined as potential targets for new classes of antibacterial compounds. Herein we present some of the data generated by efforts to validate riboswitches as drug targets and discuss some of the key unanswered questions that will determine the ultimate success of antibacterial compounds that interact with these RNAs.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Christopher W. Reid writing in Bacterial Glycomics: Current Research, Technology and Applications:

Bacteria and Archaea produce a variety of glycoconjugates such as capsular polysaccharides, lipopolysaccharides, and glycoproteins that are assembled on a polyisoprenyl-phosphate lipid in the cytoplasmic membrane. Traditional methods to analyze the membrane-associated steps of glycan biosynthesis involved the use of metabolic radio-labeling or the indirect detection of lipid-associated glycans. Recent advances in analytical biochemistry now provide the microbial glycobiologist with a number of tools for the direct detection and characterization of low abundance lipid-linked oligosaccharides. Approaches include targeted glycolipidomics strategies, such as affinity-capture capillary electrophoresis mass spectrometry and global approaches such as separation on porous graphite carbon and normal phase liquid chromatography mass spectrometry (LC-MS). These techniques provide opportunities to probe the lipid-associated steps in glycan biosynthesis in greater detail as well as provide an enabling technology for the exploitation of these pathways in glycoengineering.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Thomas Mikeska and Alexander Dobrovic writing in Epigenetics: A Reference Manual:

Methylation-sensitive high resolution melting (MS-HRM) is an inexpensive and robust closed tube screening methodology that enables rapid analysis of locus specific DNA methylation for multiple samples. MS-HRM is based on the differential melting behaviour of PCR amplification products derived from methylated and unmethylated templates after bisulfite treatment. The melting profiles of an unknown sample are compared to the melting profiles of standards with known DNA methylation levels. MS-HRM has the advantage of allowing ready distinction between homogenous and heterogeneous DNA methylation. Estimation of DNA methylation to quite low levels in a semi-quantitative manner is possible for homogeneously methylated templates where all the CpG sites are methylated. However for heterogeneous methylation, the formation of multiple heteroduplexes makes quantitation difficult. Digital MS-HRM utilises limiting dilution to amplify single templates enabling DNA methylation analysis at a single allele resolution and enables the quantitative analysis of both homogenous and heterogeneous methylation. The PCR products either generated by MS-HRM or digital MS-HRM can subsequently undergo Sanger DNA sequencing or pyrosequencing to investigate DNA methylation at individual CpG positions.

Further reading: Epigenetics: A Reference Manual

from Brian Spetman, Sarah Lueking, Brooke Roberts and Jonathan H. Dennis writing in Epigenetics: A Reference Manual:

The location and density of nucleosomes in the eukaryotic genome plays a role in regulating nuclear processes including transcription, replication, recombination, and repair. Microarray mapping of nucleosomally protected DNA has emerged as a powerful, cost-effective, high-throughput method to analyze the relationship between nucleosome position and genomic regulation. In this chapter we discuss experimental considerations such as sample preparation and microarray design. In addition, two procedures are detailed: (1) Formaldehyde Crosslink and Harvest Cells, Isolate Nuclei, and MNase cut chromatin, and (2) Isolation of mononucleosomally protected DNA and fluorescent labeling of DNA for microarray hybridization. With the information specified in this chapter, most any laboratory equipped for molecular biology with institutional or commercial access to microarray facilities should be should be able to map nucleosome position and occupancy.

Further reading: Epigenetics: A Reference Manual

A variety of specialized data resources manage the results of microbial genome data processing and interpretation at different stages. These stages correspond to different levels of microbial genome characterization. Draft and finished microbial genome data are continuously incorporated in various microbial genome data resources. Below are brief descriptions to the main microbial genome data resources.

GOLD Genomes Online Database
GOLD (Genomes Online Database) is a World Wide Web resource for comprehensive access to information regarding complete and ongoing genome projects, as well as metagenomes and metadata, around the world. GOLD was created in 1997 with the aim to (i) monitor all genome sequencing projects from instigation to completion and (ii) provide the community with a centralized database integrating diverse information related to those projects in the form of hyper-text links to disparate web-based resources. Although several different types of statistics, related to each of the data fields, can be derived from the user at any point using the search engine, the database also provides readily available graphical overviews for specific data types.

Complete and ongoing projects and their associated metadata can be accessed in GOLD through pre-computed lists and a search page. As of March 2008, GOLD contains information on more than 3613 sequencing projects, out of which 731 have been completed and their sequence data deposited in the public databases (GOLD V2.0). GOLD continues to expand with the goal of providing metadata information related to the projects and the organisms/environments towards the Minimum Information about a Genome Sequence' (MIGS) guideline.

ASAP A systematic annotation package
ASAP (a systematic annotation package for community analysis of genomes) is a relational database and has a web interface developed to store, update and distribute genome sequence data and their functional characterizations. ASAP facilitates ongoing community annotation of genomes and tracking of information as genome projects move from preliminary data collection through post sequencing functional analysis. The database includes multiple genome sequences at various stages of analysis, corresponding experimental data and access to collections of related genome resources. Its development was motivated by the need to more directly involve a greater community of researchers, with their collective expertise, in keeping the genome annotation current and to provide a synergistic link between up-to-date annotation and functional genomic data. ASAP supports three levels of users: public viewers, annotators and curators. Public viewers can readily browse updated annotation information such as for Escherichia coli K-12 strain MG1655, genome-wide transcript profiles from more than 50 microarray experiments and an extensive collection of mutant strains and associated phenotypic data. Annotators worldwide are currently using ASAP to participate in a community annotation project for the Erwinia chrysanthemi strain 3937 genome. Curation of the E. chrysanthemi genome annotation as well as those of additional published enterobacterial genomes are underway and will be publicly accessible in the near future.

CMR Comprehensive Microbial Resource
CMR (Comprehensive Microbial Resource) is a tool that allows researchers to access all the bacterial genome sequences completed to date. It contains robust annotation of all completed microbial genomes and allows for a wide variety of data retrievals. For each genome not sequenced at The Institute of Genome Research (TIGR), two kinds of annotation are displayed: the Primary annotation taken from the genome sequencing center and the TIGR annotation generated by an automated annotation process at TIGR. CMR thus allows access of all the information on all of the bacterial genomes or any subset of them. Retrievals can be based on protein properties such as molecular role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of data types.

IMG Integrated Microbial Genomes
The IMG (Integrated Microbial Genomes) system serves as a community resource for comparative analysis and annotation of all publicly available genomes from the three domains of life, in a uniquely integrated context. IMG provides tools and viewers for analyzing and annotating genomes, genes and functions, individually or in a comparative context. An increasing number of eukaryotic genomes, viruses (including phages) and plasmids have also been added to IMG in order to increase its genomic context for comparative analysis. IMG's analytical tools have been gradually generalized and enhanced in terms of their usability, analysis flow and performance. These tools allow users to focus on a subset of genes, genomes and functions of interest, and conduct analysis using summary tables, graphical viewers and various methods for comparing genes, pathways and functions across genomes.

SEED Comparative genomics research
SEED is a software environment to support early phases in building design that has been adopted for comparative genomics research. Database support in SEED allows designers to store and retrieve different design versions, alternatives and past designs that can be reused and adapted in different contexts (case-based design in the terminology of Artificial Intelligence). In addition, the database stores recurring problem specifications and typical requirements for building types or functional areas common to many buildings. The database serves also as a main means of information exchange between modules, which do not communicate design decisions directly to each other. Current literature refers to this as information modeling or product and process modeling.

Advances in microfluidics and microfabrication technologies have contributed greatly to the miniaturization of biological and chemical analytical systems, allowing the handling of low volume samples, as well as reductions in reagent consumption, waste generation, costs and assay time. Micro-total analysis systems (micro-TAS), sometimes called "lab-on-a-chip", are microfabricated devices capable of performing the functions of large analytical devices in small units. These devices are fabricated in glass, silicon or polymer materials, and integrate different functions and functionalities. Some sophisticated versions can perform sample introduction and handling (e.g., cell lysis, dilution and debris removal), separation (e.g., electrophoresis, chromatography) and detection, all conducted on the chip. It is believed that micro-TAS will be particularly valuable in DNA and protein analysis, genomics and proteomics, and diagnostics.

Miniaturized immunoassays have been performed successfully with microchips. These immunoaffinity microfluidic devices are considered promising platforms to achieve rapid and sensitive immunological detection of microbial cells. Zhu et al. described a simple approach in which sample trapping and concentration steps were integrated together with whole-cell immunoassay in a silicon-based lab-on-a-chip. The immunoassay was performed by injecting the sample solution, which contained C. parvum and G. lamblia, into a microchamber. Subsequently, a solution containing fluorescently-labelled target-specific antibodies was delivered and serves to simultaneously concentrate, trap and label the targeted cells at a trapping region. Following a wash step to remove unbound antibodies, the labelled parasites were detected by epifluorescence microscopy. Compared to conventional immunoassays, the total analysis time was reduced from 2-3 h to 2-5 min, and the total consumption of reagents was reduced 20-fold. In an alternative approach, Liu et al. injected microbeads coated with a primary antivirus antibody into a microfluidic device, which are subsequently trapped in front of a pillar-type filter region. A sample containing target virions was injected into the device and virions were captured on the surface of the microbeads. This was followed by injection of a labelling solution containing a secondary antivirus antibody labelled with QDs to allow detection by epifluorescence microscopy. In comparison to a standard ELISA performed on the same marine iridovirus, the minimal detectable concentration of the target virus was improved from 360 to 22 ng/ml, the detection time was shortened from 3 h to less than 30 min, and the amount of antibody consumed was reduced 14-fold.

Considerable effort has been directed to the development of chip-based systems for miniaturized and rapid PCR. The devices consist of a chip containing wells, channels, electrodes, filters, pumps, valves and heating devices designed for buffer and sample storage, PCR and target DNA detection. Remarkably, a polymeric microchip with a 1.7 microlitre chamber containing a thermocoupler was used to successfully amplify a 500-bp DNA fragment of lambda phage in 15 cycles, in a total amplification time of 240 seconds. By making use of a PCR microchip coupled with a capillary electrophoresis (CE) chip, it was more recently demonstrated that bacterial targets as low as 2-3 cells could be amplified within a 200-nl PCR chamber and the PCR-amplified target DNA was subsequently resolved by CE within 10 min. In order to improve PCR throughput and reduce the analysis time, multi-chamber PCR microfluidics on a single chip has been reported. Also, chip devices with optical windows have been fabricated that allows for measurement of fluorescence intensity during the thermocycling process, thus providing a miniaturized version of real-time PCR. In this regard, Cady et al. developed an integrated miniaturized real-time PCR detection device equipped with a microprocessor, pumps, thermocycler and light emitting diodes (LEDs)-based fluorescence excitation/detection. Monolithic DNA purification and real-time PCR enabled fast detection of L. monocytogenes cells (10⁴-10⁷) within 45 min.

In spite of their potentially powerful application in diagnostics and environmental monitoring, the 'complete' lab-on-a-chip still requires further development. The bottlenecks blocking the realization of a truly and highly integrated chip include sample preparation and product detection. Since the source of raw template samples is varied and the sample preparation methods are diverse, the miniaturization of conventional sample preparation and functionalities on a chip remains a challenge. As for on-chip detection, the product detection methods have not advanced as rapidly as other aspects of chip development. Consequently, miniaturized ultra-sensitive detectors are required if the sensitivity of the lab-on-a-chip devices is to be improved. Moreover, additional efforts have to be made towards the validation of the methods to demonstrate the reliability of micro-TAS systems.

from Leigh G. Monahan, Michael A. D'Elia and Elizabeth J. Harry writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

The alarming rise of antibiotic resistant bacteria in hospitals and the community has exposed a critical need for new drugs that are not merely variants of older antibiotics, but target previously unexploited proteins and pathways. The wealth of available knowledge on the process of bacterial cell division implicates the division pathway as an excellent potential target, and has aided target-driven approaches to identify novel inhibitors. In this chapter we discuss the therapeutic potential of inhibiting bacterial divison based on a strong foundation of basic research into the division mechanism and its regulation in model bacteria, and more recently, clinically relevant pathogens. In addition, we review the progress made towards identifying division inhibitors, describe new approaches for antibacterial drug development targeting division and discuss the potential challenges for the future of this exciting new area of antibacterial discovery.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

Due to advances in areas such as genomics and biotechnology, powerful methodologies have been developed to detect both specific pathogens and indicator organisms. Because many microorganisms are not easily cultured or can enter a viable but nonculturable (VBNC) state, the current methods focus on immunological or genetic characteristics to detect the presence of specific waterborne pathogenic microorganisms. Not only do these methods increase the rapidity of analysis, but they are also able to achieve a high degree of sensitivity and specificity without the need for complex cultivation and additional confirmation steps. Consequently, some of these methods permit the detection of specific culturable and/or nonculturable microorganisms within hours, instead of the days required with the traditional methods.

Detection of pathogenic organisms provides information as to the safety and public health risks associated with a given water supply; however, it often does little to define the potential sources of the contamination. Generally, because different enteric pathogens are present in the intestines of different animals, the identification of a contamination event as being either of human or animal source would provide information as to the types of pathogens that may be expected, the risk of infection and the treatment that may be required to control transmission of disease. In response, molecular techniques are being developed as means to identify the source of a contaminant. DNA fingerprinting is one tool for microbial source tracking (MST) and consists of a family of techniques that are used to identify the sources of fecal contamination in various water bodies.

The different polymorphism-based procedures are generally coupled to a PCR reaction. In the amplified ribosomal DNA restriction analysis (ARDRA) technology, PCR-amplified 16S rRNA genes are digested with restriction endonucleases and the resulting fragments separated electrophoretically. Presence or absence of the restriction site within two strains cause differences in the length of the DNA restriction fragments, and the complexity of the pattern depends upon the number of target sequences and position of restriction sites. Comparison of the generated patterns to those obtained from a database allows assignment of isolates to species or species clusters in those cases were the banding patterns are highly similar. The separated DNA fragments may also be transferred to filters for hybridization with probes specific for an organism of interest. Two other protocols for generating DNA fingerprints use a single primer to amplify fragments with PCR before examination on agarose gels. PCR amplification of repetitive extragenic palindromic sequences (Rep-PCR) takes advantage of repetitive sequences found in the microbial genome. In the randomly amplified polymorphic DNA (RAPD) or arbitrarily primed PCR technology, a short oligonucleotide primer (about 10 nucleotides), usually with random sequence that is not specific for a particular gene is used as a primer to amplify fragments. These methods yield DNA fingerprints comprised of multiple, differently sized DNA amplification products following separation by gel electrophoresis.

Detection of host-specific 16S rRNA genetic markers, using length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis, also holds promise as an effective method for characterizing a microbial population. The technique distinguishes members of mixtures of bacterial gene sequences by detecting differences in the number of base pairs in a particular gene fragment. Whereas LH-PCR separates PCR products for host-specific genetic markers based on the length of amplicons, T-RFLP uses restriction enzymes on amplified PCR products to determine unique size fragments. Specifically, in T-RFLP, rRNA target gene sequences are PCR-amplified using one or both of the primers with a fluorescent label. The amplification product(s) are then digested with appropriate restriction endonucleases and following electrophoresis of the resultant fragments using an automated DNA sequencer, a fluorescent electrophoretic profile of the digestion patterns is obtained. The use of labelled primers limits the analysis (identification) to only the terminal fragments, thus allowing the study of complex microbial communities. Moreover, the possibility of discriminating fragments with differences as small as single bases gives the method a higher resolution than gel-based profiling techniques.

One of the most promising technologies for microbial source tracking is, however, amplified fragment length polymorphism (AFLP) analysis. AFLP analysis appears to have the same taxonomic range as other fingerprinting techniques, but this technology combines several advantages of these different techniques, which in most cases results in the highest power of discrimination. This technology is based on the selective amplification of a subset of genomic restriction fragments using PCR. For AFLP, purified genomic DNA is digested with two restriction endonucleases, one with an average cutting frequency and a second one with a higher cutting frequency after which oligonucleotide adapters are ligated to the genomic DNA restriction fragments. The sequence of the adapters and the adjacent restriction site serves as oligonucleotide primer binding sites for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction fragments are added to the 3' ends of the adapter-specific PCR primers such that only a subset of the restriction fragments are recognized and amplified. The subset of amplified fragments is then analyzed by denaturing polyacrylamide gel electrophoresis to generate the fingerprint. Since relatively small amounts of DNA are digested and detection of AFLP fragments does not depend on hybridization, the AFLP analysis method is more reproducible and robust than other fingerprinting techniques and it also displays more fragments than other fingerprinting techniques.

Developments, particularly in the fields of genomics and biotechnology in the last few years, have resulted in a wide range of molecular tools, principally based on the detection of nucleic acid material and its amplification. They offer a novel, more sensitive and specific way of detecting microorganisms. They can also identify organisms that would not be detected with current culture techniques and can be used to track new pathogenic entities, including variants of otherwise harmless microorganisms. There are probably very few groups of microorganisms that have not been detected with these amplification techniques and several test kits have already been commercialized. Despite the success of these molecular methods, several barriers must be overcome before they can be used to routinely assess water quality and the microbiological safety of drinking water. The relationship between detection by molecular approaches and the subsequent viability or infectivity of waterborne enteric pathogens remains a concern. In addition, methods used for purifying and concentrating the target microbes and their nucleic acids from water, so that they are free of contaminants that may interfere with the analysis, need further improvement, consolidation and simplification. Further research is also needed to develop and refine the prototype protocols into collaboratively tested methods that could be used routinely and expeditiously to evaluate the microbiological safety of water.

Owing to recent advances in nanoscience and nanotechnology, various different nanomaterials and devices have been developed that show great promise for diagnostic applications. Subsequently, many different nanotechnology-based diagnostic systems have been reported in the literature and many of these have the potential to become the next generation of diagnostic tools. Moreover, microfluidic chip-based systems such as the lab-on-a-chip technology should have a significant impact on environmental microbial monitoring by permitting detection and identification of targets within minutes at the sampling site with a sensitivity level of a single cell. However, some technical and practical problems need to be solved before their full potential can be realized. These include tight control over the synthesis and functionalization of nanomaterials, as small variations can change their properties and behaviour in diagnostic methods. Also, their implementation into routine functional devices remains a challenge, and note should be taken that many of the diagnostic systems must still be taken from proof-of-concept and evaluated with environmental samples. In this regard, some of the challenges that need to be resolved, in addition to those highlighted above, include sample processing, detection of multiple agents in a single sample, and improving the sensitivity and selectivity of the assays for application to complex environmental samples.

from Jennifer E. Cropley and Catherine M. Suter writing in Epigenetics: A Reference Manual:

Epigenetic states are faithfully inherited through mitotic cell division, but are generally cleared and reset on passage through the mammalian germline. But this clearing of epigenetic marks is not always complete, leading to transgenerational inheritance of epigenotype. Transgenerational epigenetic inheritance has been demonstrated in several organisms, including mammals, and has been most comprehensively studied in mouse strains carrying variants of the agouti (A^vy) and axin (Axin^Fu) alleles. The most prominent feature of transgenerational epigenetic inheritance is its non-Mendelian nature: not all offspring that inherit the genetic locus also inherit the parental epigenetic state. Transgenerational epigenetic inheritance is emerging as an important facet of mammalian biology. It may underlie the etiology of human diseases that display complex patterns of inheritance, including diabetes, mental illnesses and autoimmune diseases. As variable epigenetic states can be inherited on an invariant genotype, epigenetic variation may provide a substrate for Darwinian selection that is independent of genetic variation.

Further reading: Epigenetics: A Reference Manual

Nanocantilevers, which are typically made of silicon, silicon nitride or silicon oxide, require only minute changes in compressive or tensile surface stress on either their upper surface or lower surface to cause measurable deflection of the cantilever, and are capable of converting biomolecular recognition reactions into micromechanical motion. Consequently, nanocantilevers offer an opportunity for the development of highly sensitive, miniature and label-free detection systems.

Direct, label-free detection of DNA typically involves the use of silicon cantilevers with a thin gold coating on the top surface that is functionalized with thiolated capture DNA strands. Binding of the capture DNA strands with the introduced target DNA causes deflection of the cantilever, which can be measured accurately using optical detection methods. Using this approach, Fritz et al. demonstrated the hybridization of complementary oligonucleotides with a detection limit of 10 nM and showed that a single mismatch between two 12-mer oligonucleotides is clearly detectable. Similarly promising results were obtained by Hansen et al. with a 10-mer oligonucleotide.

In recent years, cantilever immunosensors have been developed to detect bacteria and viruses. Typically, the devices detect the additional mass loading that results from the interaction between specific antibodies, immobilized on the surface of the cantilever, and antigens on the cell membrane surface. In an early experiment, a cantilever biosensor was constructed and used to detect E. coli O157:H7, following immersion of the cantilever in a suspension containing 106-109 cells/ml. The detection of 16 E. coli O157:H7 cells were demonstrated and no frequency shifts were observed when buffer alone or buffer containing S. enterica serovar Typhimurium was incubated with the cantilevers. A magnetoelastic cantilever immunosensor has been developed that uses a magnetic field to induce oscillation of the sensor. The sensor surface is coated with antibodies to permit specific capture of the desired target agent after which alkaline phosphatase-labelled antibodies to the target are added to amplify the signal, thereby increasing the total mass on the sensor. The sensor was tested with E. coli O157:H7 and a sensitivity of 102 cells/ml were reported. More recently, resonant cantilever biosensors have been developed for detection of Listeria innocua and vaccinia virus, and the detection of a single L. innocua cell and vaccinia virus particle were demonstrated.

Despite being in its early days, cantilevers provide an opportunity for label-free, real-time measurements in fluids in a single-step reaction, and can potentially serve as a powerful platform for sensitive, multiplexed detection of biomolecules. Although cantilevers can be microfabricated by standard low-cost silicon technology, leading to a decrease in production costs and allowing the possibility of integrating multiple functional devices onto the same platform, some challenges must be overcome before cantilever array sensors can come into widespread use. These challenges relate especially to methods for detecting nanoscale motion and the development of immobilization techniques that can efficiently transduce the stress involved in biochemical interaction to the cantilever substrate.

from John F. Kelly and Ken F. Jarrell writing in Bacterial Glycomics: Current Research, Technology and Applications:

Archaea are single-celled microorganisms that are sufficiently distinct as to constitute a third domain of life. It has been known for some time that Archaea express glycoproteins. However, research to understand the nature of their glycan modifications, the pathways used to produce them as well as their role in archaeal biology has lagged behind similar efforts in Eukarya and Bacteria, at least until recently. Here we describe in some detail the efforts made to determine the composition and structure of archaeal glycan posttranslational modifications and to elucidate their biosynthetic pathways.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Robert J. Goldstone, Roman Popat, Matthew P. Fletcher, Shanika A. Crusz and Stephen P. Diggle writing in Microbial Biofilms: Current Research and Applications:

It is now well recognised that populations of bacteria from many Gram-positive and Gram-negative species cooperate and communicate to perform diverse social behaviours including swarming, toxin production and biofilm formation. Communication between bacterial cells involves the production and detection of diffusible signal molecules and has become commonly known as quorum sensing (QS). In addition, an evolutionary perspective on QS illuminates important phenomena which help in understanding the prevalence and diversity of QS phenotypes and strategies under various conditions. The research fields of QS and biofilm formation often overlap with a number of studies demonstrating that QS is an important regulatory mechanism of biofilm formation in a variety of bacterial species. However in contrast, there are conflicting reports, demonstrating that QS appears to play a minimal role in the development of biofilms. Our aim in this review is to highlight the key findings with respect to QS and the subsequent impact on biofilm formation. We also discuss QS and cooperation in the context of social evolution and how this may impact on the development and maintenance of microbial biofilms.

Further reading: Microbial Biofilms: Current Research and Applications

from Catherine Bougault, Sabine Hediger and Jean-Pierre Simorre writing in Bacterial Glycomics: Current Research, Technology and Applications:

Liquid-state NMR is traditionally used to provide fine chemical and structural information on soluble fragments, but is limited to biomolecules in the fast rotational tumbling regime. On the other hand, solid-state NMR, unlimited by the molecular size, is extensively applied to polymers, but provides a spectral resolution that is hampered by orientational heterogeneity. This chapter analyzes the potential of high-resolution solid-state NMR in providing chemical, structural and dynamics information on whole bacteria or bacterial cell envelopes in lyophilized as well as in fully-hydrated samples. The first section addresses bacterial strain typing issues and discusses the choice of adequate nuclear probes, the concomitant requirements for efficient isotope labeling schemes and the NMR methods used to characterize the chemical composition of peptidoglycan, teichoic acids, lipopolysaccharides or mycolic acids in bacteria. The second section describes the principles used to characterize molecular interactions of proteins, ions, antibiotic/antimicrobial molecules with the bacterial cell wall by NMR. The third and last section gives an overview of the contribution of solid-state NMR to characterize the cell wall structure and dynamics, and covers the different techniques used to extract specific structural information as well as global mobility.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Allan Matte, Ian C. Schoenhofen, Traian Sulea, Miroslaw Cygler and N. Martin Young writing in Bacterial Glycomics: Current Research, Technology and Applications:

Many hexose sugars in bacteria undergo a variety of modifications, including oxidation/reduction, amination and acetylation, as part of biosynthesis into their final biologically-active forms. Enzymes that catalyze these reactions normally utilize nucleotide-linked sugar substrates, utilizing the nucleotide as an 'ancient handle' to bind and orient the sugar within the enzymes' active site. We and others have focused efforts on elucidating structure-function relationships for a subset of such biosynthetic enzymes, those associated with the synthesis of trideoxy-diacetamidohexoses, and the nonulsonate sugars subsequently derived from them. With structural information combined with site-directed mutagenesis, enzymatic analysis and molecular modeling, these studies have been essential to understanding the chemistry of how these enzymes bind their substrates and effect catalysis. Enzymes having different folds, such as N-acetyltransferases, can utilize different scaffolds to attain the same 4-acetamido-sugar product. In the case of dehydratases/epimerases and aminotransferases, the enzymes have a conserved structure, but utilize subtle differences within their active site to confer substrate binding and the nature of the final product. These studies depict the structural relationships between these enzymes, while at the same time high-lighting important differences that are beginning to reveal their function at the molecular level.

Further reading: Bacterial Glycomics: Current Research, Technology and Applications

from Venkatachalam Lakshmanan, Amutha Sampath Kumar and Harsh P. Bais writing in Microbial Biofilms: Current Research and Applications:

Microorganisms have historically been studied as planktonic or free-swimming cells, but most exist as sessile communities attached to surfaces, in multicellular assemblies known as biofilms. In the process of coping with both the pathogenic and beneficial interactions, the rhizosphere of plant roots encourages formation of sessile communities that begins with the attachment of free-floating microorganisms to a surface. Certain bacteria such as plant growth promoting rhizobacteria not only induce plant growth but also protect plants from soil-borne pathogens in a process known as biocontrol. Contrastingly, other rhizobacteria in a biofilm matrix may cause pathogenesis in plants. Although research suggests that biofilm formation on plants is associated with biological control and pathogenic response, little is known about how plants regulate this association. The scope of this chapter is restricted to biofilm-forming bacteria and their interactions with terrestrial plants, specifically emphasizing recent work. After an overview of documented interactions between bacteria and plant tissues, we examine some of the more prominent mechanisms of biofilm formation on and around plant surfaces.

Further reading: Microbial Biofilms: Current Research and Applications

from Mohammad Ali Faghihi and Claes Wahlestedt writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

One prominent and complex class of regulatory RNAs is natural antisense transcripts. Natural antisense transcripts (also known as antisense RNAs or antisense transcripts) are RNA molecules that are transcribed from the opposite DNA strand and are often overlapping in part with mRNA of conventional sense genes. Transcription of antisense RNA, similar to sense RNA, is tissue and cell line specific. Indeed a large fraction of antisense transcripts is expressed in specific regions of the brain, supporting involvement of these regulatory RNAs in sophisticated brain functions as well as in complex neurological disorders. Recent research on natural antisense transcripts, including several large-scale expression-profiling studies, has conclusively established the existence of antisense transcripts in eukaryotic genomes. In fact, the consensus opinion is that natural antisense transcripts, most of which represent non-protein-coding RNAs, transcribed abundantly in the mammalian genome. However, there are many unanswered questions that still exist concerning antisense transcripts biological functions and their heterogeneous mode of actions in various cells. For instance, what fraction of antisense RNAs may have functional significance, and how many different regulatory mechanisms may exist for these RNA molecules? Natural antisense transcripts appear to be utilizing various cellular pathways, but it is still not clear which intrinsic properties of antisense RNA molecules or extrinsic features, such as protein interactions, cellular and developmental context are decisive for the selection of any given pathway. How is the expression of these non-protein-coding RNAs regulated in various cells, and what are the extrinsic factors that affect the transcription of antisense RNAs? Considering tissue- and cell type-specific expression patterns of antisense RNAs and their heterogeneous proposed functions, natural antisense transcripts appear to be a heterogeneous group of regulatory RNAs with a wide variety of biological roles.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Enrique M. Muro and Miguel A. Andrade writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

Pseudogenes are genome loci that look like genes but have sequences apparently prevented to produce any functional product due to genetic defects. However, recent advances in the field of molecular biology urge the revisiting of this definition. In this chapter we will discuss some of those advances. There is experimental and computational evidence of some biological function arising from pseudogene transcription but this evidence is not easy to find. Accordingly, not that many studies have been published on the topic. It seems that if there is pseudogene transcript functionality, it arises in certain tissues, and in certain conditions, with much more specificity than gene expression. Some of this complexity relates to the fact that this function involves non-coding RNAs (ncRNAs), a molecular entity for which novel tools and biological paradigms are still being worked out. A particular type of ncRNAs are Natural Antisense Transcripts (NATs) and these have a special relevance for the study of pseudogene functionality for reasons that we will discuss.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Salah Mahmoudi, Anna Vilborg and Marianne Farnebo writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

The p53 tumor suppressor is arguably the most important player in preventing tumor formation and progression. p53 mutations are found with high frequency (about 50%) in human tumors, however virtually all tumors have inactivated the p53 pathway in some fashion.The p53 protein functions as a transcription factor with a crucial role in orchestrating the cellular stress response. p53 can be activated by a large variety of stress factors, and reacts by triggering an appropriate response, the nature of which will vary with the triggering factor and the cellular background. The most classical outcomes of p53 activation are cell cycle arrest and apoptosis, and it is generally believed that these two, together with senescence, irreversible cell cycle arrest, are the p53 responses with greatest impact on preventing tumor formation2. However, it has lately become evident that p53 is involved in many other processes in addition to these. p53 can promote several forms of DNA repair, thus contributing to maintaining genomic integrity. Further, p53 may promote differentiation of stem and progenitor cells into more specialized cell types, and can also prevent self-renewal of stem cells. Paradoxically, p53 can also induce genes involved in promoting survival. One of these genes is p21, while being a classical cell cycle arrest-inducing p53 target, p21 antagonizes apoptosis. The list of pro-survival p53 targets also includes several antioxidant genes and genes involved in metabolism. The logic behind this surprising p53 function may be that the elimination of every cell ever exposed to any kind of stress is not desirable, while protecting us from getting cancer, it would lead to tissue degeneration. In addition to its crucial role in cancer, p53 has been implicated in several other diseases, generally diseases connected to excessive cell death. These include diabetes, cell death after ischemia, and various neurodegenerative diseases such as Huntington, Parkinson, and Alzheimer. Since p53 is able to eliminate cells through apoptosis and senescence and to induce differentiation, thus reducing stem cell populations, p53 has also been suggested to promote aging. Initial studies in mice expressing constitutively active p53 also seemed to confirm this hypothesis, However, subsequent mice models carrying an extra copy of p53 but under control of its normal regulatory elements demonstrated that properly controlled p53 did not induce aging. Instead it actually promoted longevity, largely by preventing tumorigenesis. Due to its critical function in deciding on life or death for the cell, strict regulation of p53 levels and activity is crucial. p53 protein levels are kept under tight control by multiple mechanisms. Further, p53 mRNA stability and translation is subject to regulation by a number of factors, including long non-coding RNA.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Alka Saxena and Piero Carninci writing in Epigenetics: A Reference Manual:

In the past decade, we have become acquainted with an entire new world of fine regulatory control within cells governed by non-coding RNAs. Although we have not completely explored this world, we know from a few well studied examples that not only gene dosage but protein function also, is fine tuned by non-coding RNAs. It appears that proper cell function is largely dependent on non-coding RNAs, many of which were invisible to us until the advent of high throughput sequencing and tiling array technology. The realization, that transcripts with no open reading frames are biologically active molecules with multiple functions including regulation of the expression of coding RNA, shifts the power from proteins to non-coding RNAs as key modulators of cellular function. In this review, we discuss the various classes of non coding RNAs and the mechanisms employed by them to achieve this status, with a particular focus on their ability to induce epigenetic modifications.

Further reading: Epigenetics: A Reference Manual

Epigenetics: A Reference Manual Edited by: Jeffrey M. Craig and Nicholas C. Wong ISBN: 978-1-904455-88-2 Publisher: Caister Academic Press Publication Date: September 2011 Cover: hardback

read more ... ]]>http://www.caister.com/molecular-biology-blog/2011/07/epigenetics-book-available-very-soon.htmlhttp://www.caister.com/molecular-biology-blog/2011/07/epigenetics-book-available-very-soon.htmlMon, 04 Jul 2011 07:20:30 GMTNon-coding RNAs, Epigenomics and Complexity in Human Cells

from Fabricio F. Costa writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

During the past two decades, new technologies in molecular biology and human genetics have enabled the discovery of different types of non-coding RNAs. Non-coding RNAs are RNA transcripts that have no apparent protein product. These molecules have been grouped in different classes such as microRNAs, small RNAs and long RNAs (lncRNAs) according to their size and function. LncRNAs have been strongly associated to epigenetic mechanisms in different cell types. Conceivably, they have been described as an essential part of the human epigenome. In this chapter, examples of lncRNAs and their function will be presented. A historical perspective on the impact of lncRNAs in epigenetic mechanisms, human disease and evolution will be also discussed.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Nicholas C. Wong writing in Epigenetics: A Reference Manual:

Biological experiments are rapidly moving into the information age with the explosion of data generated from rapidly evolving technologies. Microarrays can interrogate many thousands to millions of loci within any one sample, while massively parallel sequencing platforms can essentially measure the entire genome. Keeping up with this rapid pace of data accumulation has required the development of online tools for the processing, analysis and annotation of data generated from large numbers of microarrays or next generation sequencing (NGS) experiments. I will cover a selected range of tools available to the biological researcher, from online discussion forums and blogs, to curated databases that store the data and associated annotations. I will then cover viewer tools that enable visualisation the annotations and finally review tools for assay design for follow up validation of NGS and microarray analyses. I will pay particular attention to DNA methylation and provide the reader with an insight into what is available online for the epigenetics researcher aiming to make sense of epigenomic data. As a disclaimer, this chapter is by no means a comprehensive list of available epigenetics resources, which are constantly being updated and expanded through the Internet and just a mouse click through Google away.

Further reading: Epigenetics: A Reference Manual

from Mario A. Arteaga-Vazquez and Ana E. Dorantes-Acosta writing in Epigenetics: A Reference Manual:

Paramutation is a fascinating phenomenon in which epigenetic information can be transmitted through trans-interactions between one allele of a gene to another allele or between homologous DNA sequences that establishes a state of gene expression that is heritable for generations. Paramutation was discovered in maize and similar phenomena have been described in other plants, fungi and animals. In this chapter, we describe several classic plant paramutation systems and discuss recent advances that implicate a role for RNA and a number of components of an RNA-based transcriptional silencing pathway on paramutation.

Further reading: Epigenetics: A Reference Manual

from Andreas Werner writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

The complexity of an organism is driven by a positive balance between creative and destructive forces in the process of evolution. Small non-protein-coding RNAs play an instrumental role in both regulating gene expression (creative influence) as well as suppressing selfish genetic elements (defensive role). There are three main groups of small RNAs including microRNAs, endogenous siRNAs and piRNAs. MicroRNAs represent the most important gene regulatory small RNAs and act by tuning the expression level of eukaryotic mRNAs. Interestingly, endogenous siRNAs as well as piRNAs apparently serve both regulatory and defensive purposes. They suppress the expression of repetitive DNA elements but also influence the expression of protein coding genes. For endo-siRNAs, intriguing roles in epigenetic regulation are emerging. The multiple tasks of small RNAs are in line with a role as drivers of organismal complexity.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Haruaki Tomioka writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease to cause morbidity and death. However, the development of new drugs for the treatment and prophylaxis of TB has been slow. Therefore, novel types of antituberculous drugs, which act on the unique drug targets in MTB pathogens, particularly the drug targts related to the establishment of mycobacterial dormancy in host's macrophages, are urgently needed. In this context, it should be noted that current anti-TB drugs mostly target the metabolic reactions and proteins which are essential for the growth of MTB in extracellular milieus. It may also be promising to develop another type of drug that exerts an inhibitory action against bacterial virulence factors which cross talk and interfer with signaling pathways of MTB-infected host immunocompetent cells such as lymphocytes, macrophages and NK cells, thereby changing the intracelluar milieus favorable to intramacrophage survival and growth of infected bacilli. In this chapter, I will describe recent approaches to identify and establish novel potential drug targets in MTB, especially those related to mycobacterial dormancy and cross-talk with cellular signaling pathways.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Thomas Bjarnsholt, Tim Tolker-Nielsen and Michael Givskov writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

It is now evident that bacteria assume the biofilm mode of growth during chronic infections. The important hallmarks of biofilm infections are development of local inflammations, extreme tolerance to the action of conventional antimicrobial agents and an almost infinite capacity to evade the host defense systems in particular innate immunity. In the biofilm mode, bacteria use cell to cell communication termed quorum-sensing (QS) to coordinate expression of virulence, tolerance towards a number of antimicrobial agents and shielding against the host defense system. Chemical biology approaches may allow for the development of new treatment strategies focusing on interference with cell to cell communication with the aim of primarily disabling expression of virulence, immune shielding and antibiotic tolerance. Here we present our experience with screening and testing small molecule chemistry for N-acyl homoserine lactone dependent QS inhibition. In addition we present our thoughts with respect to advantages and potential limitations of the intervention strategies described.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Risini D. Weeratna and Michael J. McCluskie writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Infectious disease remains one of the main causes of mortality and morbidity worldwide. Vaccination has had the greatest impact of any medical intervention technique in controlling infectious diseases. Most notably, eradication of smallpox was achieved through concerted and rigorous mass vaccination programs, and the incidence of diphtheria, pertussis, polio and other childhood diseases have been significantly reduced through routine infant immunization. However, with a move away from whole-killed vaccines for safety reasons, a key challenge in realizing the full potential of vaccination has been the lack of immunogenicity of many novel vaccines especially in certain populations such as the elderly and the immunocompromised. Adjuvants are a key component in enhancing immunogenicity of vaccines. Furthermore, adjuvants can play a vital role in facilitating the induction of the appropriate type of immunity that is required to either prevent, such as in prophylactic vaccines, or to treat, such as in therapeutic vaccines. Therefore, careful consideration of the choice of adjuvants becomes quintessential for developing an effective vaccine. This chapter focuses on the importance of choosing the correct adjuvant or adjuvant combination to induce the appropriate immune responses to control the target pathogen.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Ronald J. Quinn and Jeffrey E. Janso writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Natural products and derivatized natural products, produced mainly by actinomycetes, have been one of the most successful sources of drugs used to treat and cure infectious diseases. However, many bacteria have quickly become resistant to the majority of antibiotics in use today prompting an urgent need to discover new classes of antibacterial compounds. The goal of this chapter is to summarize some of the recent advances that favorably position natural products drug discovery in the quest to discover new antibacterial agents. This includes new sources of biodiversity such as plants and the oceans as well as the overlooked potential within common soil-derived actinomycetes. Other encouraging advancements include: (1) the development of new culturing techniques, which have enabled the isolation of microbes that were once thought to be uncultivable, (2) the impact of sequencing technology and bioinformatics that have made strain dereplication more reliable and revealed that actinomycete genomes encode far more secondary metabolite gene clusters than originally thought and (3) the use of innovative methods to express and exploit these orphan biosynthetic pathways. Finally, the ability to dereplicate, isolate and elucidate the structure of natural products from less and less sample quantity will also be discussed.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from L. Silvia Munoz-Price, and John P. Quinn writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

We summarize the epidemiology, clinical presentation, and current treatment options for the most clinically relevant multidrug resistant Gram-positive and Gram-negative organisms. Additionally, we describe the challenges faced by pharmaceutical companies within the antimicrobial research and development field, especially the disproportion between the degree of investment (both monetary and time) required and the relatively small profit antimicrobial agents bring. Finally, some potential solutions for the lack of antimicrobial agents are discussed. These include more widespread use of the Orphan Drug Act, patent extensions, and the Biomedical Advanced Research and Development Authority (BARDA).

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Juilee Thakar and Eric T. Harvill writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Integrated pharmacokinetic-pharmacodynamic models are commonly used to study the in vivo dynamics of antimicrobial agents and bacterial pathogens. These models are extremely useful for understanding the properties of antimicrobial agents such as absorption, transport, rate of binding, etc. However, they fail to consider within-host aspects of the infectious process that are likely to affect the bacterial-host interactions. For example, immune-mediated mechanisms to contain bacteria or limit their access to nutrients can also affect the access of a drug to its bacterial target. Alternatively, pathogens have various strategies to sequester themselves from host immune mechanisms that can also affect the access of therapeutic agents. The search for new antibacterial agents that will be effective in vivo can be substantially informed by an understanding of the within-host dynamics of bacterial pathogens. Mathematical modeling of immune responses can assist in this process by providing new predictions, by offering mechanistic understanding and by revealing the gaps in our current understanding. Such models are based on experiments that reveal the components of the immune system that play important roles during infections. But knowing the components alone usually provides only a static picture of bacterium-host interactions. Mathematical models aim to use the information obtained from experiments to construct the interactions and dependencies between various components. Thus mathematical models offer a mechanistic understanding of the interplay between various immunological processes and simulations of these models give a dynamic view of the entire process. In this chapter we will first provide an overview of pharmacokinetic and pharmacodynamic models followed by a review of some of the immunological processes involved in bacterial infections which are generally ignored in pharmacodynamic models but are likely to affect access or activity of treatments. We will then discuss the development of mathematical models by different approaches. We will end the chapter by exploring implications of these models in the discovery of new antibacterial agents.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Mariano Alló and Alberto R. Kornblihtt writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

Since the discovery of splicing in 1977, alternative splicing was seen for more than two decades as an interesting mechanism to generate protein diversity but with limited genome-wide influence because it was thought to affect only 20% of mammalian genes. The sequencing of the human and other mammals‰Ûª genomes in the early 2000's together with more recent high throughput analyses of splicing isoforms generated a renewed interest in alternative splicing. We know now that alternative splicing affects more than 90% of human genes, that normal and pathological cell differentiation not only depends on differential gene transcription but also on alternative splicing patterns, that mutations that either create or abolish alternative splicing regulatory sequences, named splicing enhancers and silencers, are a widespread source of human disease and that alternative splicing factors can be misregulated in cancer. The relationship between splicing and non-coding RNAs has emerged recently in the middle of an avalanche of papers showing how chromatin context could affect splicing choices. The convergence of these previously unrelated areas (non-coding RNAs, chromatin and splicing) has presented a novel and intriguing scenario that will be covered in this chapter, starting with a brief overview of transcription and splicing introducing the understanding of how these processes could be modulated by external factors. Then we will focus on our work using non-coding small RNAs (ncsRNAs) to regulate alternative splicing in human cells. Finally, we will discuss the evidence supporting the potential activity of endogenous non-coding RNAs as modulators of alternative splicing.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Heather B. Felise, Toni Kline & Samuel I. Miller writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Antibiotic resistance is threatening our ability to treat bacterial diseases. Scientific development to define new antibacterial targets, including those that inhibit microbial virulence rather than target essential cellular functions, is required to develop the therapeutics of the future. In this chapter we will discuss the feasibility of Gram-negative secretion systems as therapeutic targets, provide a synopsis of current research on the identification and development of secretion inhibitors, and discuss their possible future utility as antimicrobial agents.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Hongchang Cui writing in Epigenetics: A Reference Manual:

Cell-fate specification and stem-cell renewal are fundamental processes in the development of multicellular organisms. In both animals and plants, a key role for transcription factors in these processes has been established, but an accumulating body of evidence indicates that epigenetic regulation also plays a critical role. Once regarded as stable marks, all epigenetic modifications, including DNA and histone methylation, are now known to be reversible, and a cohort of enzymes that add or erase epigenetic marks has been identified. Throughout the life of an organism, the epigenome is dynamically modified, leading in turn to transcriptional changes and ultimately cell-fate specification. Here, I review the recent literature that has shaped this view. Plants are unique among complex organisms in having the ability to generate a whole new organism from a single differentiated cell. How plant cells retain this amazing capability while maintaining their cell identity is a mystery. I introduce a model system for study of cell-fate specification, stem-cell renewal, and reprogramming.

Further reading: Epigenetics: A Reference Manual

from Adam M. Nelson and Vincent B. Young writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Recent technological advances have expanded the tools available for study of the indigenous human microbiota. One of the early limitations in this field was the difficulty in recovering most residents of the community via standard culture-based methods. Many residents of the flora are anaerobic or microoxic, require specific nutrients, or are dependant on microbe-microbe/microbe-host interactions that are difficult to replicate in vitro, thus making their cultivation difficult. Naturally, the easiest species to grow in the laboratory have been the best studied. However, these cultivatable species are only a fraction of the total population of the microbiota. This chapter will introduce both the culture and non-culture based techniques being used to look deeper into the population structure both on a temporal and spatial scale. It will also discuss how disruptions (including those mediated by the administration of antibiotics) of the microbiota can produce changes in human health, and outline ongoing efforts by the National Institutes of Health and international investigators to study the indigenous microbiota.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Marnie E. Blewitt and Linden J. Gearing writing in Epigenetics: A Reference Manual:

X chromosome inactivation is the method of dosage compensation that has evolved to equalise expression of X-linked genes between female (XX) and male (XY) mammals. In somatic cells only one X chromosome is active; the second X in female cells is silenced early during embryonic development, a process that involves the co-ordination of multiple levels of epigenetic regulation to ensure stable chromosome-wide silencing. In this chapter we shall focus on the molecular mechanisms involved in X chromosome inactivation, discuss how the epigenetic marks are believed to elicit stable transcriptional silencing, and why X inactivation represents an excellent model system for studying epigenetic regulation in mammals.

Further reading: Epigenetics: A Reference Manual

Theodoros N. Rampias, Emmanuel G. Fragoulis and Diamantis C. Sideris

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3 ́ RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3 ́UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3 ́ RACE procedures.
Recommended reading

from Daniel H. Kim and Jeannie T. Lee writing in Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection:

X-chromosome inactivation provides a model for discovering the still emerging functions of noncoding RNAs, which exhibit diverse roles in regulating the epigenome. Combining recent insights into X-chromosome noncoding RNAs with the advent of next-generation sequencing technologies promises many new discoveries in interrogating the functions of noncoding RNAs at the genomic level. Global RNA-protein interaction information is readily available through utilizing RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq), which has recently uncovered the diversity of the noncoding Polycomb transcriptome. In this chapter, we discuss noncoding RNAs of the X-inactivation center that have provided unique insights into RNA molecules as molecular switches, guides and tethers to the epigenome, and key participants in diverse regulatory pathways, including RNA interference and Polycomb silencing.

Further reading: Non-coding RNAs and Epigenetic Regulation of Gene Expression: Drivers of Natural Selection

from Jason Gill and Ryland F. Young III writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

A bacteriophage, or "phage", is a virus that infects bacteria. This chapter is aimed at assessing the record and potential of the use of phage and phage-derived molecules in antibacterial therapeutics and prophylactics. Unlike other areas of current biomedicine, phage therapy has a long history that pre-dates even the basics of modern biology, and even the development of phage biology itself. Thus it is important to reflect on the historical record to establish a context before considering the more recent literature and, finally, the prospects and obstacles facing phage therapy at the current time. In addition, although the study of phage was vibrant through the mid 1970s, the last decades of the 20^th and the first decade of the 21^st centuries witnessed a drastic contraction in the number of phage biology laboratories. This has led now to an odd situation where interest and activity in phage research are outstripping the available expertise. Accordingly, a section of this chapter is devoted to a summary of the fundamental characteristics of bacteriophage that would be important to the prospective phage therapist. Next, we present a review and metareview of the recent phage therapy literature and then summarize the current practices in the field. Finally, we consider the future, in terms of what should be done, according to our perspective. Please note that throughout this text, we define terminology for elements and concepts important to phage biology and its practical applications. We have done this in an overt attempt to simplify the text, but in some cases we admit to promoting what we think is better and less confusing terminology than that currently in general use. To this end, a glossary is provided at the end of the chapter.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Danny Rangasamy writing in Epigenetics: A Reference Manual:

The histone variants of the H2A family are highly conserved in mammals, playing critical roles in regulating many nuclear processes by altering chromatin structure. One of the key H2A variants, H2A.X, marks DNA damage, facilitating the recruitment of DNA repair proteins to restore genomic integrity. Another variant, H2A.Z, plays an important role in both gene activation and repression. A high level of H2A.Z expression is ubiquitously detected in many cancers and is significantly associated with cellular proliferation and genomic instability. This review summarizes the current understanding of these variants and their functions, as well as their links to cancer development. Furthermore, the significance of dysfunction of these variants is highlighted with respect to their potential as biomarkers and as new targets for anticancer therapy.

Further reading: Epigenetics: A Reference Manual

dsDNA dyes are commonplace in the molecular biology laboratory. Although ethidium bromide was first used in real-time PCR, SYBR Green I is by far the most common dye in real-time PCR today. Introduced along with the LightCycler, it is more fluorescent than ethidium bromide and is easily excited at the same wavelength as fluorescein. Most real-time PCR is performed with dsDNA dyes for reasons of cost and convenience. Any PCR can be monitored with SYBR Green I. However, because dsDNA dyes are generic, there is a risk of non-specific detection of alternative PCR products. This risk can be partly eliminated by acquiring fluorescence at a temperature where only the desired product is double-stranded. Melting analysis can also differentiate between specific and non-specific products (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

from Justin A. Fincher and Jonathan H. Dennis writing in Epigenetics: A Reference Manual:

DNA in eukaryotes is efficiently and compactly organized into chromatin, the fundamental subunit of which is the nucleosome: approximately 150 bp of DNA spooled 1.65 times around a histone octamer. The location and density of nucleosomes play a role in regulating nuclear processes including transcription, replication, recombination, and repair. Mechanisms acting in trans, like ATP-dependent remodelers and cellular memory complexes, as well as in cis features intrinsic to the DNA sequence itself regulate the location and density of nucleosomes. Here, we review the three cis acting DNA sequence features that affect the distribution of nucleosomes: (1) two frameworks defining the relationship between the histone octamer and the underlying DNA sequence (nucleosome occupancy and nucleosome position, then statistical positioning and a nucleosome positioning code), (2) the organization of DNA into the nucleosome core particle, and (3) specific DNA sequence features and DNA templates that promote or inhibit the formation of nucleosomes. We close by describing three computational algorithms trained on DNA sequence that have been used to predict nucleosome position and density. In summary, we hope to draw attention to multiple aspects of DNA sequence that specify organization of sequence into nucleosomes and influence the distribution of nucleosomes in eukaryotic genomes.

Further reading: Epigenetics: A Reference Manual

Significant advances in the detection of sequence-specific nucleic acid hybridization have been achieved using microarrays. Microarrays are glass microslides or nylon membranes containing a high density of immobilized nucleic acids (genomic DNA, cDNA or oligonucleotides) in an ordered two-dimensional matrix. Microarrays can be prepared by synthesizing DNA in situ on a glass surface using combinational chemistry or by robotic microdeposition of cDNAs (0.5- to 2-kb) amplified by PCR. The sample DNA, usually bound to a fluorescent or enzyme label, is exposed to the microarray and hybridizes with the target sequences. The detection of the probe-target hybrid at each spot on the array is achieved either by direct fluorescence scanning or enzyme-mediated detection yielding a semi-quantitative result. Advantages of DNA microarray technology, as compared to other techniques, include the small size of the array allowing for a higher sensitivity, the ability to simultaneously detect diverse individual sequences in complex DNA samples, and the capacity to do comparative analysis of a large number of samples. Indeed, PCR-microarrays have been used to measure relative concentrations of microbes from water, to characterize microbial communities from environmental samples and to detect bacterial pathogens from a variety of sources, including water. However, obstacles such as sample size, matrix-associated inhibitors, nonspecific binding and cross-hybridization must be overcome before microarrays can be used generally for the detection and differentiation of pathogens in environmental samples. Moreover, microarrays cannot distinguish between nonviable, culturable and viable but nonculturable (VBNC) cells since DNA can persist for long periods of time after the death of cells. The ability to discriminate between living and nonliving cells is important in order to interpret the risk associated with the detection of pathogenic microbes (especially in environmental samples). The use of mRNA, which is highly labile with a short half-life, or the use of highly expressed targets may be selected to provide sensitive analyses.

from Samson Mani and Zdenko Herceg writing in Epigenetics: A Reference Manual:

DNA methylation is an important regulator of gene transcription and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing, and the genes with high levels of 5-methylcytosine in their promoter region are transcriptionally silent. DNA methylation is essential during embryonic development, and in somatic cells, patterns of DNA methylation are generally transmitted to daughter cells with a high fidelity. Aberrant DNA methylation patterns have been associated with a large number of human malignancies and found in two distinct forms: hypermethylation and hypomethylation compared to normal tissue. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumour suppressor genes. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms. This chapter will focus on DNA methylation as the major epigenetic mechanism involved in normal biological processes and abnormal events leading to cancer development. It will also focus on the interaction between DNA methylation and other epigenetic mechanisms.

Further reading: Epigenetics: A Reference Manual

Traditionally, prediction of the presence of human enteric pathogens in water has been achieved by monitoring for established microbial "indicators" of fecal pollution. Not necessarily pathogenic themselves, fecal coliforms, total coliforms, E. coli, enterococci and bacteriophages are all examples of organisms that when present are viewed as predictive of the potential presence of enteric pathogens, since they have the same fecal source as the pathogenic organisms. Tests for coliform bacteria are standardized and relatively easy and inexpensive to use. Consequently, they are more rapidly administered than tests determining the presence of individual pathogenic microorganisms in water. Despite being successful in predicting possible health risks in many circumstances, there are many flaws in using microbial indicators. Research has established an inability of many of these indicators to predict the presence of disease-causing viruses (such as hepatitis A and E, coxsackie viruses, echoviruses, adenoviruses and Norwalk viruses), indigenous bacteria (such as Legionella and Helicobacter), as well as parasites (such as Cryptosporidium and Giardia).

Since it would appear that conventional detection methods do not adequately assess the risk of waterborne disease, the need for powerful new tools for the detection of pathogenic microorganisms in water is becoming increasingly more important. Although several different molecular methodologies are available, these have only recently been applied in the field of water science and technology. Moreover, the rapid progress of nanotechnology and advanced nanomaterials production offers significant opportunities not only for the detection and remediation of a broad range of environmental contaminants, but also for the development of new diagnostic assays that may serve as an appealing alternative to current molecular diagnostic techniques.

from Robert G.K. Donald and Annaliesa S. Anderson writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Prophylactic anti-bacterial vaccines have been responsible for a drastic reduction in global bacterial diseases. Older vaccines made from attenuated whole cells or lysates have been largely replaced by less reactogenic acellular vaccines made with purified components, including capsular polysaccharides and their conjugates to protein carriers, inactivated toxins (toxoids) and proteins. Examples of vaccines in each category are reviewed to illustrate underlying strategies and associated technological advances such as polysaccharide conjugation and recombinant protein expression. In addition, progress and the current status in the development of new vaccines to prevent diseases caused by N. meningitidis serogroup B, S. aureus and C. difficle is summarized. Future progress will likely bring to the clinic passive immunotherapies based on monoclonal antibodies and new adjuvants, especially for use in vaccines against intracellular pathogens.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

By measuring time, temperature, and fluorescence throughout PCR, real-time 3-dimensional spirals can be acquired and plotted. Software on commercial instruments usually only present selected data. For example, qPCR experiments only acquire fluorescence at one temperature each cycle. Typical melting analysis only acquires fluorescence from one melting curve at the end of amplification. Much more data is available during PCR, and it is likely that this additional data will find further use in the years to come.

Homogeneous monitoring of PCR is the method of choice for gene expression quantification and closed-tube genotyping. As a "gold standard", it has evolved from early conception to present-day reality. Future improvements will be focused on reducing cost and complexity (high resolution melting), decreasing reaction volumes (microfluidic PCR) and increasing throughput and sensitivity (digital PCR). These approaches will allow homogeneous monitoring of PCR to continue its evolution as a useful tool for many years to come (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

High Resolution Melting Analysis

Labeled primers became unnecessary with the development of saturating dsDNA dyes that could detect heteroduplexes throughout an amplicon. Single base genotyping within small amplicons required only two PCR primers and became the simplest method of genotyping. Because heterozygotes were easily identified by a change in melting curve shape, variant scanning was also enabled. Unlike other scanning techniques, high resolution melting does not require any physical processing or separations. It has been applied to cancer, many human genetic disorders and has been recently reviewed. In clinical diagnostics, greater sequence detail can be obtained if necessary with unlabeled probes or snapback primers (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Unlabeled probe genotyping requires only two standard primers and an unlabeled probe complementary to the sequence of interest. Unlabeled probes are generally designed to be 20-35 bases long and can match either the wild-type or variant sequence. Snapback primers incorporate the unlabeled probe as a 5'-extension on one of the primers. At low temperature the probe element snaps back onto its complementary sequence to form an intramolecular hairpin. With both unlabeled probes and snapback primers, different genotypes result in varying duplex stabilities which are easily resolved by high resolution melting. When unlabeled probes or snapback primers are used for genotyping, two melting transitions are generally observed, one for the full-length amplicon and the other for the probed region. This feature enables simultaneous variant scanning and genotyping in the same PCR reaction (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Microfluidic PCR

However, despite wide interest in microfluidic PCR, progress has slowed from a number of setbacks. Sample and reagent adsorption onto the reaction vessel surfaces can inhibit PCR and increase the risk of carryover contamination, due to the large surface area-to-volume ratios used. Solutions for sample adsorption have been explored with differing success. PCR efficiency in microfluidic PCR is often compromised, and the samples used for demonstration are often less complex targets such as plasmids, bacteria or previously amplified products at high concentrations.

There are two widely used designs for microfluidic PCR; stationary well and continuous flow. Stationary wells do not move the sample and operate in much the same way as traditional thermal cyclers. Both the sample and the device itself are heated/cooled through specific temperatures for PCR. The thermal mass is still large and thus more traditional cycling times are generally employed to perform the PCR. However, a matrix of microwells, mixing all combinations of X samples and Y targets can be obtained by microfluidics, greatly simplifying reaction preparation.

Continuous-flow microfluidic PCR has some advantages over PCR performed in stationary wells. Namely, by moving the sample through fixed temperature zones, this system can achieve faster cycling times due to the fact that only the sample needs to be heated and not the entire system. A comprehensive review of the various designs and techniques involved in microfluidic PCR is available (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Digital PCR

The total reaction volume of the many wells required for digital PCR make 96- or even 384-well plates unwieldy for high-throughput sample analysis. However, digital PCR performed on microfluidic PCR devices has been used for single-copy DNA droplet PCR, aneuploidy detection and absolute quantification of point variants.

Digital PCR improves detection specificity and sensitivity in samples with a large background of wild-type alleles compared to variant alleles and is the ultimate in allele quantification. The reduced cost associated with microfluidic devices may eventually make single-step, highly parallel individual PCR reactions for digital PCR affordable (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).]]>http://www.caister.com/molecular-biology-blog/2011/04/current-and-future-trends-in-pcr.htmlhttp://www.caister.com/molecular-biology-blog/2011/04/current-and-future-trends-in-pcr.htmlWed, 13 Apr 2011 04:00:18 GMTPCR TechnologyReal-Time PCRfrom Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

After the introduction of hydrolysis and hybridization probes, several other probe designs were adapted to, or created for, real-time PCR. Some of these probes increase in fluorescence when their conformation changes with hybridization and may function by hybridization and/or hydrolysis mechanisms depending on the reaction conditions. Hairpin probes, or "molecular beacons" can be monitored in real-time. These probes have a central sequence complementary to the DNA target and flanking ends complementary to each other. This configuration creates a hairpin at low temperatures. At higher temperatures in the presence of target, the probe hybridizes preferentially to the target. One end of the probe is labeled with a fluorophore and the other with a quencher so that when hybridized to the target, fluorescence increases. Hairpin probes use quenchers that release transferred energy as heat rather than light (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Hairpins can also be attached to the 5'-end of a PCR primer to generate self-probing amplicons during PCR. A fluorophore/quencher pair in the hairpin stem is linked to the primer with a blocking agent that prevents PCR read-through. The loop of the hairpin is complementary to the extension product of the primer so that once extension occurs, intramolecular hybridization separates the fluorophore/quencher pair and a larger hairpin is formed. This intra-molecular hybridization of self-probing amplicons is faster than the intermolecular hybridization of other probes. Hairpin primers can also be made without a blocking agent, allowing PCR read-through and incorporation of the stem-loop into the product. During PCR, the quencher/reporter pair is separated and fluorescence increases.

from Anne K. Voss and Tim Thomas writing in Epigenetics: A Reference Manual:

A histone (H3-H4)₂ tetramer flanked by two H2A-H2B heterodimers form the core protein structure, around which DNA is wrapped. DNA and the histone octamer together form the smallest chromatin particle, the nucleosome. How intimately the DNA associates with the core histones and how tightly the nucleosomes are packed with each other is determined by a key post-translational modification of the histone proteins, namely acetylation. Histone acetylation was first discovered in the early 1960s. After a dearth of progress, due to technical limitations, our knowledge of histone acetylation has exploded in the last fifteen years. Enzymes that catalyse acetylation of histones, the histone acetyltransferases, have been discovered, proteins associated with these have been identified and their preferences for specific histone residues have been determined. Importantly, we are gaining a better understanding of the relevance of histone acetylation in health and disease through the discovery of genetic mutations underlying human diseases in loci encoding histone acetyltransferases (HATs) and through examination of mouse strains deficient in specific histone acetyltransferases. Here we discuss the principles of histone acetyltransferase biology.

Further reading: Epigenetics: A Reference Manual

Understanding the regulatory mechanisms should allow the examination of engineering pathways with pre-determined expression patterns (i.e. expression is activated by a given compound or in a specific environmental or physiological condition). Metabolic pathways have evolved to execute their function efficiently, while tolerating perturbations, such as changes in environmental parameters or in the physiological status of the cell. Below we describe some of the databases and programs for integrated analyses of metabolic pathways.

KEGG
KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database of biological systems that integrates genomic, chemical and systemic functional information. KEGG provides a reference knowledge base for linking genomes to life through the process of PATHWAY mapping. The PATHWAY database contains information about conserved sub-pathways (or pathway motifs), which are often encoded by positionally coupled genes on the chromosome and which are especially useful in predicting gene functions. The genomic information is stored in the GENES database, which is a collection of gene catalogs for all the completely sequenced genomes and some partial genomes with up-to-date annotation of gene functions. A third database in KEGG is LIGAND that includes information about chemical compounds, enzyme molecules and enzymatic reactions. In addition, KEGG provides a reference knowledge base for linking genomes to the environment, such as for the analysis of drug-target relationships, through the process of BRITE mapping. KEGG BRITE is an ontology database representing functional hierarchies of various biological objects, including molecules, cells, organisms, diseases and drugs, as well as relationships among them. Additionally, the KEGG resource is being expanded to suit the needs for practical applications. KEGG DRUG contains all approved drugs in the US and Japan, and KEGG DISEASE is a new database linking disease genes, pathways, drugs and diagnostic markers.

KEGG provides Java graphics tools for browsing genome maps, comparing two genome maps, manipulating expression maps, as well as including computational tools for sequence comparison, graph comparison and path computation.

BioCyc
The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB offers a wealth of genomic and metabolic information on certain microorganisms, including P. aeruginosa and S. cerevisiae. Each database provides information on a microorganism's annotated genome, on the biochemical reaction(s) that each gene product catalyses and on the organism's metabolic pathways, predicted from its annotated genome by a program called PathoLogic. The information from each database is comprehensive and complex. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms.

MetaCyc
MetaCyc is a universal database of metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are curated from the primary scientific literature, and the small-molecule metabolic pathways are experimentally determined. Each reaction in a MetaCyc pathway is annotated with one or more well-characterized enzymes. Because MetaCyc contains only experimentally elucidated knowledge, it provides a uniquely high-quality resource for metabolic pathways and enzymes. MetaCyc stores pathways involved in both primary metabolism and secondary metabolism. MetaCyc also stores compounds, proteins, protein complexes and genes associated with these pathways. It is extensively linked to other biological databases containing protein and nucleic-acid sequence data, bibliographic data and protein structures. MetaCyc also contains objects for the genes that encode many enzymes within the DB. While it does not contains primary sequence data, MetaCyc does contain links to external sequence databases.

EcoCyc
EcoCyc is a bioinformatics database that describes the genome and the biochemical machinery of E. coli K-12 MG1655. The long-term goal of this project is to describe the molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists, and for biologists who work with related microorganisms. EcoCyc contains the complete genome sequence of E. coli, and describes the nucleotide position and function of every E. coli gene. The annotation of the Escherichia coli K-12 genome in the EcoCyc database is one of the most accurate, complete and multidimensional genome annotations. EcoCyc information was derived from 15 000 publications. The database contains extensive descriptions of E. coli cellular networks, describing its metabolic, transport and transcriptional regulatory processes. Database queries to EcoCyc survey the global properties of E. coli cellular networks and illuminate the extent of information gaps for E. coli, such as dead-end metabolites. EcoCyc provides a genome browser with novel properties, and a novel interactive display of transcriptional regulatory networks.

The advent of DNA microarray technology has greatly expanded our ability to monitor changes in the abundance of transcripts. Such a development has been a milestone in several areas of microbiology. In clinical microbiology, microarrays are used for microorganism detection and identification and gene-expression analysis. DNA microarrays have allowed us to monitor the effects of pathogens on host-cell gene expression in a much greater depth and on a significantly broader scale than previous single gene studies. The results generated by these studies are complex, and few systematic studies have been carried out to compare results among studies. Comparative transcriptomics - whole genome mRNA transcript profiling using microarrays.

Whole-genome microarrays from fully sequenced genomes are a powerful platform for identifying differences in gene content between organisms and for studying gene expression dynamics. The generation of messenger RNA expression profiles is referred to as transcriptomics, as these are based on the process of transcription. Given the inhospitable in vivo and the varied ex vivo environments encountered by most microbial pathogens, transcriptome analysis holds particular promise for identifying and determining the functions of differentially regulated, virulence associated genes. The basic principle of this technique involves extracting the mRNA expressed under a range of environmental conditions and hybridizing these sequences to a high-density gridded microarray of the DNA content of an organism. Such high-throughput analysis allows massive parallel gene expression and gene discovery studies to be undertaken. DNA microarray analysis will complement other technologies such as in vivo expression technology and differential fluorescence analysis to identify and investigate which bacterial genes are differentially expressed in the host.

The application of DNA microarrays to microbial pathogens
The study of the complete set of genes expressed and modified in a cell is an important and rapidly evolving discipline that is readily applicable to microbial pathogens. For example, strains of Staphylococcus aureus resistant to the antibiotic vancomycin present a potentially serious public-health problem. In the case, the Gaasterland group described the development of a multi strain S. aureus microarray. Pairwise comparisons of the available genomes of strains of S. aureus have revealed considerable variation in gene content across the epidemiological landscape. They identified changes in protein-coding potentials that correlate with antibiotic resistance by measuring differences in gene expression in vancomycin-sensitive and vancomycin-resistant pairs of S. aureus isolates. Philip Butcher's group used microarrays to help understand the complex pathophysiology of Mycobacterium tuberculosis infection. Besides discussing some methodological aspects of microarray work, They focused on the use of M. tuberculosis microarrays to investigate the intracellular lifestyle of this organism and its interaction with host macrophages. In the future, it would be exciting to integrate results from in vitro work like this with results from in vivo microarray work on mammalian hosts to provide a whole-genomic view of host-pathogen interactions.

from Sebastian Lunke and Assam El-Osta writing in Epigenetics: A Reference Manual:

With the advances in traditional genetics failing to provide causal genes for many complex diseases, the focus of research is shifting towards determining the importance of gene-environment interactions, more specifically epigenetic regulation. Paramount to answering this question is the knowledge of which transcription factors bind to what sequence, as well as a detailed understanding of how the transcriptional state of a genetic sequence is epigenetically distinguished. The chromatin immuno-precipitation (ChIP) strategy has proven to be a powerful tool to investigate these mechanisms, but has been limited for a long time to single locus analysis. The recent emergence of next-generation sequencing (NGS) technology however has revolutionized the field of epigenomics and for the first time enabled unbiased genome-wide analysis of ChIPed DNA. Here we provide a detailed discussion and protocol on how to best perform ChIP for NGS analysis.

Further reading: Epigenetics: A Reference Manual

from Arturo Casadevall writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

At the beginning of the 21^st century the therapeutic paradigm for the treatment of infectious diseases can be summarized by three words: kill the bug. In other words, the overwhelming majority of therapeutic interventions against microbial diseases are designed to help the host by damaging the microbe directly and/or interfering with its ability to replicate in tissue (Casadevall, 2006). This strategy has been termed the second age of antimicrobial therapy and was preceded by the era of serum therapy, which differed in the fundamental manner that serum was primarily an immunotherapeutic agent than enhanced host defenses (Casadevall, 2006). First and second age therapeutics differed in other ways including the chemistry of the therapeutic agent, their specificity and the form of manufacturing (Table 1). Second age therapeutics have been were tremendously successful and brought numerous drugs to the market that have saved countless lives. However, there are major trends at work that have significantly reduced the overall efficacy of second age therapeutics including widespread antimicrobial resistance, the emergence of new pathogenic microbes for which there are few drugs available and an epidemic of immunocompromised hosts where antimicrobial therapy is often less effective. Microbe-targeting strategies are limited in that they neglect the host; consequently, there are very few treatment strategies that aim to achieve a therapeutic outcome by enhancing host defenses. Microbe-targeting strategies include both microbe-specific and -non-specific drugs, each of which can put tremendous selection pressure on microbes that often result in the emergence of resistance. Non-specific microbe-targeting strategies have the additional problem that they can select for resistance in non-targeted microbes and their effects on host flora can have a variety of unintended deleterious consequences on host homeostasis. This chapter will consider these strategies in light of their historical development and analyze the advantages and disadvantages of specific and non-specific antimicrobial strategies.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Emma L. Northrop and Lee H. Wong writing in Epigenetics: A Reference Manual:

In eukaryotes, each chromosome has one centromere and its ends are protected by telomeres. The centromere is a specialized chromosomal locus that directs kinetochore assembly and provides the site for microtubule attachment, allowing accurate chromosome segregation during cell division. Despite the critical role centromeres play, centromeric DNA sequences are highly variable and not conserved between species. Increasing evidence, including the discovery of functional neocentromeres, suggests that centromere identity and function is epigenetically defined through the formation of a specialised chromatin structure. This chapter reviews recent studies addressing the structural and functional characterisation of centromere chromatin, its assembly and propagation during cell division. Telomeres are specialized nucleoprotein complexes that protect the chromosome ends from degradation. In recent years, it has become increasingly clear that heterochromatic marks at telomeres act as negative epigenetic regulators of telomere elongation, repress recombination events at the telomere and are critical for maintaining telomere structural integrity. Recent research reporting telomeres being transcribed by RNA polymerase II to give rise to TERRA RNA, open up an additional level of regulation at the telomere. This chapter will discuss the links between the epigenetic status of telomeres, telomere function and telomere-length regulation, and the implications on cellular reprogramming, aging and cancer.

Further reading: Epigenetics: A Reference Manual

Since their discovery, carbon nanotubes (CNTs) have attracted great attention as nanoscale building blocks for micro- and nanodevices. CNTs can be divided essentially into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) based on the principle of hybridized carbon atom layers in the walls of CNTs. Whereas SWCNTs have diameters ranging from 0.3 to 3 nm, the MWCNTs are composed of a concentric arrangement of many cylinders and can reach diameters of up to 100 nm. CNTs are considered to be ideal for use in biosensors for detecting individual biomolecules and other biological agents. Not only do they have high surface-to-volume and surface-to-weight ratios, but they are also conducting, act as electrodes, and can be derivatized with functional groups that allow immobilization of biomolecules.

The use of nano-size electrodes based on CNTs has the potential to greatly improve the sensitivity in recognizing DNA hybridization events. Li et al. developed DNA microarrays containing sensing pads constructed from MWCNTs and built into a matrix within a silicon nitride template. The upper (open) ends of the tubes act as nanoelectrodes and are functionalized with ssDNA probes. Target DNA that hybridizes to the ssDNA probe on the ends of the electrically conductive MWCNTs is detected using an electrochemical method that relies on guanine oxidation. The hybridization of less than a few attomoles of oligonucleotide targets was demonstrated. In an alternative approach, CNTs coated with alkaline phosphatase enzymes was used for the detection of amplified DNA. This assay employs a magnetic microparticle modified with oligonucleotides that are complementary to one-half of the target DNA sequence, and alkaline phosphatase-coated carbon nanotubes that are modified with oligonucleotides that are complementary to the other half of the target DNA sequence. Binding of the target DNA promotes the formation of a magnetic microparticle/target/carbon nanotube sandwich, which is magnetically separated from the assay medium. After separation, the enzyme substrate alpha-naphthyl phosphate is added to the mixture, resulting in formation of alpha-naphthol product that is ultimately detected at a CNT-modified electrode via chronopotentiometric stripping. This method detected target DNA at concentrations as low as 54 aM.

In addition to their potential in recognizing DNA hybridization events, the use of CNTs displaying ligands for the capturing or recognition of bacterial pathogens has recently been described. The solubilization of SWCNTs via fictionalization with derivatized galactose has been reported and it was subsequently shown that the nanotube-bound galactose could serve as polyvalent ligands that strongly interacted with receptors on E. coli O157:H7, resulting in significant cell agglutination. This work has subsequently been extended to the preparation of immuno-carbon tubes. For this purpose, SWCNTs and MWCNTs are functionalized with bovine serum albumin (BSA) to attain aqueous solubility and then further conjugated with an E. coli O157:H7-specific antibody to form immuno-carbon tubes. Limited quantitative data was provided, but the results suggest that the immuno-carbon tubes are capable of sensitively capturing the target bacteria.

While CNTs currently are not as easily functionalized as QDs or nanoparticles, they offer the distinct advantage of rapid, real-time detection and may thus become viable options as nanostructured biodiagnostic devices. In addition to challenges at the fabrication level (e.g., production of pure and uncontaminated nanotubes is costly, continuous growth of defect-free CNTs to macroscopic lengths is difficult to obtain and dispersion of CNTs onto a polymer matrix is very difficult), another important issue related to the use of CNTs is their toxicity. Although results suggest that chemically modifying CNTs can reduce their cytotoxicity to a certain extent, more research is required to address the effect of CNTs on biological systems, as well as information related to safety issues.

from Miina Ollikainen writing in Epigenetics: A Reference Manual:

A large variety of methods to measure DNA methylation have been developed and used extensively over the last twenty years. These have been based on selective restriction digestion of methylated DNA, the capture of methylated DNA by methyl binding proteins or antibodies, or bisulphite conversion of DNA. However, all restriction enzyme based methods are dependent on available restriction sites for methylation-specific restriction enzymes and therefore cannot be used to analyse every CpG site in the genome. Although immunoprecipitation methods are not sequence specific, they are unable to provide methylation data at a single-base resolution. Therefore, both of these approaches are limited in their applicability. On the other hand, bisulphite conversion based methods allow methylation studies directed to any CpG site in the genome. Sodium bisulphite treatment of DNA converts all unmethylated cytosines into uracil while methylated cytosines remain unchanged, thus transferring an epigenetic difference into a measureable genetic difference. A variety of downstream methods such as Polymerase Chain Reaction (PCR), sequencing, Single Nucleotide Polymorphism (SNP) genotyping and mass spectrometry can be coupled with bisulphite conversion. This chapter provides an overview of some of these methods, concentrating on bisulphite sequencing, methylation-specific PCR, pyrosequencing, MassARRAY EpiTYPER and Infinium HumanMethylation27 BeadChip. Considerations for assay selection and detailed protocols for each method are presented in the end of this chapter.

Further reading: Epigenetics: A Reference Manual

from Jay Fitzgerald, Younjoo Lee and Chaitan Khosla writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Since the discovery of penicillin, the development of anti-infective drugs has been a central theme in the pharmaceutical industry through much of the 20^th century. However, the pace of developing new anti-infective agents has precipitously declined in the past two decades. The main reason for this change is an economic one - whereas the technical and regulatory risks associated with the development of a new broad-spectrum antibiotic are deemed unacceptably high, the financial returns derived from a targeted (narrow-spectrum) antibiotic are unattractive to the pharmaceutical industry. Meanwhile, the need for new anti-infective agents continues to be as urgent as ever. New business models are called for, ones that are grounded in the possibilities and realities of 21st century technologies for antibiotic discovery and development. This chapter discusses, using four selected examples, the opportunities for harnessing modern biosynthetic insights and engineering methods to discover new antibiotics.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Mark D. Robinson, Bryan Beresford-Smith, Anthony Kaspi and I. Haviv writing in Epigenetics: A Reference Manual:

Since biological phenotype and differentiation is regulated partially through CpG DNA methylation, and this mark is relatively easy to measure, genome-wide profiling of methylation landscape is a popular tool in epigenetic research. The burden then falls on bioinformatics to provide normalization, quality control, and interpretation, while adopting to the vast number of versatile methods to interrogate methylation profiles. While creating a relational report of the results from either of these methods is critical for data to cross over from lab to lab, and from research to diagnostic and translation purposes, the methods are each introducing their unique bias to the data. Ideally, one would hope all labs would adopt a single method, but since each method offers a unique advantage, such as price, sensitivity, or confidence, one has to overcome the disparity hurdle via bioinformatic tools. Thus identifying the methodological biases of each technique, and the way to compensate for those in the process of generating a universal CpG methylation prediction on a genome region is the ultimate goal we are describing here. Follow up of CpG methylation with other read outs, such as impact on gene expression or coincidence with locations in the genome, where allelic variation is associated with the investigated phenotype, are also key to proper interpretation of the results.

Further reading: Epigenetics: A Reference Manual

from Alex J. O'Neill writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Antibiotic resistance is conferred by heritable genetic determinants that enable a bacterium to grow and cause disease in the presence of therapeutically-achievable concentrations of the corresponding antibiotic. However, bacteria may also become refractory to the killing action of antibacterial agents in ways that do not fit this definition, and which are collectively referred to here as 'antibiotic survival'. These phenomena, which include drug indifference, tolerance, persistence, and the recalcitrance of biofilms to antibacterial agents, are believed to play a central role in antibacterial treatment failure. In addition, they can extend the duration of treatment required to resolve bacterial infections, and facilitate the emergence of acquired antibiotic resistance. This chapter will provide an overview of the different types of antibiotic survival, and will discuss chemotherapeutic approaches to minimising or overcoming the problems that they present to effective antibacterial treatment.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

Before thermostable polymerases were used in PCR, thermal cyclers were unwieldy instruments with integrated fluidics to add fresh enzyme after each denaturation. Taq polymerase greatly reduced the engineering complexity of thermal cyclers, requiring only temperature cycling but not liquid handling. It did not take long before a variety of thermal cycling solutions appeared. Instruments progressed rapidly from laboratory oddities to mainstream commodities. Some early homemade examples changed the temperature of stationary reactions with flowing water or robotically transferred samples between constant temperature water baths (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). However, water has some drawbacks. Due to its large thermal mass a great amount of energy and time is required to heat or cool water to a specific temperature. In contrast, air has a very low thermal mass and was used in some early systems (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Many thermal cyclers now use Peltier elements and metal blocks for heating and cooling.

Today, PCR hardware and reagents are commonplace in research and diagnostic laboratories. The instruments have evolved to fill a variety of batch size and time-to-result needs. Thermal cycling concerns now focus on issues of speed, temperature uniformity, sample volume and increased throughput. Many thermal cycling solutions, heat-stable polymerases, and commercial PCR master mixes that include all components except primers and template DNA are available commercially.

A big step in PCR automation was connecting the amplification and detection stages to control PCR product contamination. Laboratories can be plagued by false positive results if products from a prior reaction find their way into a future reaction with the same primers. This contamination is usually controlled by separating pre- and post-amplification processes and careful attention to reaction preparation (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Another solution is to automate both amplification and detection in a closed-vessel system, eliminating PCR product exposure to the environment. The best solution is to amplify and analyze at the same time by real-time PCR and/or melting analysis.

from Bret R. Sellman and C. Ken Stover writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Prior to the use of antibiotics, antibody (or serum) therapy was used with some success to treat bacterial infections. Antibiotics almost completely replaced the use of antibody therapies for bacterial disease with few exceptions. Based upon the information available at the time, this was an obvious progression given the broader spectrum activity of antibiotics. Antibiotics revolutionized medicine and the approach to treating infectious disease. In addition to their broad spectrum, they exhibited few side-effects relative to the potential for serum sickness (following the administration of equine immune serum) and they were inexpensive. But bacterial resistance to antibiotics became evident in the decades to follow, and we are now faced with a shortage of effective antibiotics and a need for alternative approaches to stand-alone antibiotic therapy. One such approach which could supplement antibiotic use, thereby removing some of the selective pressure from antibiotics, is monoclonal antibody therapy or prophylaxis. Recent advances in monoclonal antibody technology and discovery strategies and the ability to make a fully human antibody have led to the marketing of ~30 recombinant antibodies and Fc fusion proteins to treat a variety of human diseases. Although this technology has yet to yield an antibacterial product, many clinical and preclinical programs are underway to explore varied and novel approaches to monoclonal antibody-based anti-infectives.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

from Alita A. Miller and Paul F. Miller writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

A global public health crisis due to antibiotic resistance may be imminent. Several organizations are working to mitigate the lack of new, effective drugs either in development or in the clinic by proposing strategies for re-investment in antibacterial research. Although it is imperative that regulatory issues be resolved and strategic policies be put in place, it is equally important to define the scientific path required to address this crisis. The goal of this textbook, therefore, is to offer new ways of thinking about antibiotics and technical solutions for the resistance problems we face. By summarizing innovative new concepts and approaches from leading experts around the world, we hope to enable the implementation of the re-investment strategies that are so urgently needed.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

The rapid accumulation of bacterial genome sequences has opened up a new field of research, that of comparative genomics. Interpretation of raw DNA sequence data involves the identification and annotation of genes, proteins, and regulatory and/or metabolic pathways. Therefore, there is a natural shift towards the creation of tools for viewing and manipulating data in a comparative genomics context. In addition, genome annotations need to be reprocessed on a regular basis to take into account the newly characterized functions of genes. Furthermore, large-scale functional analyses generate additional data that contribute to the interpretation of genomic data. These considerations are driving the research community to think about how to manage public collections of genomes in novel ways. One role of bioinformatics is to assist biologists in the extraction of biological knowledge from this flood of data. Consequently, software designed for the analyses and functional annotation of a single genome have evolved to tools for comparative genomics, detecting the relatively conserved information across many genomes simultaneously. Here we introduce several popular tools for bacterial genome annotation and comparative genomics.

from Audrey N. Schuetz and Yi-Wei Tang writing in Emerging Trends in Antibacterial Discovery: Answering the Call to Arms:

Despite the rising numbers of multidrug resistant pathogens, and their continuously emerging resistance patterns, few novel antibacterial agents have been approved or released recently. In order to combat this problem, efforts are being made to extend the utility of existing antibiotics as long as possible, while attempting to develop new drugs. The clinical practice of evidence-based therapy, based on diagnosing early and narrowing antimicrobial coverage, with timely administration of an antibiotic, may help alleviate the problem. Diagnostic procedures optimized for accuracy and turn-around time further improve patient therapy. We review techniques currently in use in diagnostic microbiology, such as direct microscopic examination, rapid biochemical and antigen testing, microorganism culture, serologic diagnosis, and a variety of molecular diagnostic techniques. In addition, we introduce various emerging diagnostic techniques, which show promise in their application towards a more exact antibacterial practice. Such emerging technologies include ultra high-throughput sequencing, microarray science, quantum dots, PCR electrospray ionization mass spectrometry, atomic force microscopy, and carbon nanotubes. Point-of-care testing devices are also reviewed. As diagnostic methods have changed over the years, the novel applications of these technologies hold promise in their rapidity and accuracy, while showing potential application in drug target testing and drug discovery.

Further reading: Emerging Trends in Antibacterial Discovery: Answering the Call to Arms

]]>http://www.caister.com/molecular-biology-blog/2011/02/conference-update.htmlhttp://www.caister.com/molecular-biology-blog/2011/02/conference-update.htmlWed, 09 Feb 2011 12:22:33 GMT5-methylcytosine As a Modification in RNA

from Jeffrey E. Squires and Thomas Preiss writing in Epigenetics: A Reference Manual:

A wealth of nucleobase and ribose modifications have been identified in multiple types of RNA including tRNAs, rRNAs, mRNAs, and small regulatory RNAs. Among them, 5-methylcytosine (m⁵C) has been detected in rRNAs, tRNAs, and early reports have indicated its presence in mRNAs. Well established as an epigenetic mark in DNA, the prevalence and function of m⁵C in RNA is either incompletely explored (tRNA, rRNA) or virtually unknown (mRNA, other noncoding RNA). Two eukaryotic m⁵C RNA methyltransferases have been identified; however, their substrate specificity and biological roles are incompletely understood. With recent advances in bisulfite sequencing of RNA, comprehensive analyses to determine the occurrence and functions of m⁵C in the transcriptome now appear feasible. In this chapter, we summarise the current knowledge in this field, focussing primarily on eukaryotic transcriptomes.

Further reading: Epigenetics: A Reference Manual

Quantum dots
Quantum dots (QDs) are colloidal, luminescent inorganic nanocrystals with unique photochemical properties, which include high quantum yields, large extinction coefficients, pronounced photostability, as well as broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. A typical QD has a diameter of 2-8 nm and is usually composed of a core consisting of a semiconductor material, such as CdSe, enclosed in a shell of another semiconductor material with a larger spectral band-gap, such as ZnS. The shell prevents the surface quenching of excitons in the emissive core and thus increases the photostability and quantum yield for emission. Since QDs are usually synthesized from organometallic precursors and salts, they have no intrinsic aqueous solubility. Consequently, the native coordinating organic ligands on the surface of the QDs must either be exchanged or functionalized with a ligand that can impart both solubility and bioconjugation sites, if desired. Strategies for attaching biorecognition molecules to QDs comprise of direct chemical coupling to a functional group displayed on the QD surface or by non-covalent self-assembly in which proteins are engineered to express positively charged domains that interact through electrostatic attractions with the negative surface of modified QDs.

Although functionalized QDs have been used to detect complementary target nucleic acid sequences in chip-based assays and in fluorescent in situ hybridization (FISH) assays, are QDs increasingly being used in immunoassay detection. QD-based immunological assays have been applied to the detection of different bacterial and protozoan pathogens. For these immunoassays, the QDs are conjugated to organism-specific antibodies, or, alternatively, biotinylated organism-specific antibodies are used that are subsequently detected using a QD-streptavidin bioconjugate. These approaches have been used successfully for the detection of Cryptosporidium parvum (43 oocysts in spiked reservoir water), Giardia lamblia (1-5ˆó10³ cysts) and Escherichia coli O157:H7 (2ˆó10⁷ cells). Moreover, QDs appear to be especially suited for multiplex immunoassays. As a demonstration of the potential of QDs in multiplexed immunoassay formats, the simultaneous detection of E. coli O157:H7 and Salmonella enterica serovar Typhimurium bacteria (10⁴ cells in 2 h), and of C. parvum and G. lamblia (5ˆó10³ cysts) in environmental water samples, using different coloured QDs as immunoassay labels, has been reported.

QDs have also been applied in flow cytometry because of their broad absorption spectra and narrow size-tunable photoluminescent emission spectra. The broad absorption band enables semiconductor QDs to simplify and improve the detection of multiple bacterial targets in flow cytometry samples. Each organic dye needs a separate excitation source resulting in multiple excitation sources and complicated experimental setups when monitoring more than one organism. In comparison is only a single excitation source in the UV range needed to excite all the visible colours of CdSe QDs. QDs have been used to simultaneously enumerate pathogenic E. coli O157:H7 and harmless E. coli DH5alpha in water. Cross spectral talk of QDs are also drastically lower than the organic dyes traditionally used in flow cytometry due to their narrow emission spectra. Flow cytometric measurements of QDs have been compared with the widely used fluorochrome, fluorescein isothiocyanate (FITC). The minimum fluorophore concentration for detection was a 100-fold lower when paramagnetic beads were labeled with QDs.

Despite their capability for single molecule and multiplexed detection, it is, however, unlikely that QDs will completely replace traditional organic fluorophores as biological labels. Some of the challenges that have yet to be overcome include financial costs, since QDs are expensive in comparison to organic dyes and there is an initial financial investment required regarding instruments optimized for use with QDs. Non-specific binding and aggregation are two factors that can lower QD fluorescence as in the case of antibody stained Cryptosporidium oocysts. Also, QDs are an order of magnitude larger than organic dyes and thus the extent to which their presence perturbs the recognition event being detected, must be determined. This is particularly important when multiplex assays are desired, since labelling several biomolecules with QDs of different sizes could result in varying degrees of perturbation due to the large differences in the QD sizes. In contrast, most organic dyes are of similar size in spite of their large differences in absorption/emission characteristics.

Advances in nanotechnology and nanosciences are having a significant impact on the field of diagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. The promise of increasing sensitivity and speed, and reduced cost and labour makes nanodiagnostics an appealing alternative to current molecular diagnostic techniques. New synthesis, fabrication and characterization methods for nanomaterials, which have dimensions that range from 1 to 100 nm, have evolved to a point that deliberate modulation of their size, shape and composition is possible, thereby allowing control of their properties. Along with these advances has come the ability to tailor their binding affinities for various biomolecules (proteins, nucleic acids and microbial pathogens) through surface modification and engineering. Each of these capabilities allows the design of materials that can potentially be implemented into new biodiagnostic assays that can compete favourably with the current molecular diagnostic methodologies. In this section, the most promising nanomaterials and their use in the detection of nucleic acids and microbial pathogens will be highlighted.

Nanowires have been explored as signal transduction motifs in the electrical detection of DNA, proteins and microbial pathogens. Nanowire sensors operate on the basis that the change in chemical potential accompanying a target binding event can act as a field-effect gate upon the nanowire, thereby changing its conductance. The ideal nanowire sensor is a lightly doped, high-aspect ratio, single-crystal nanowire with a diameter between 10 and 20 nm. Recently, detailed protocols for fabrication of silicon nanowire devices, including their covalent modification with biorecognition molecules, have been described.

Real-time, label-free detection of DNA has been demonstrated by Hahm and Lieber using silicon nanowires functionalized with peptide nucleic acid (PNA). The conductance of the PNA-functionalized silicon nanowire bridging two electrodes was measured in the presence of target DNA and mutant DNA with three consecutive base deletions. Introduction of target DNA into the assay caused a rapid and immediate change in conductance, while the effect of mutant DNA was negligible. Furthermore, the conductance changes scale with target concentration, and target DNA at concentrations as low as 10 fM were detected.

Semiconducting silicon nanowires have also been used for the electrical detection of viruses in solutions. Patolsky et al. interfaced nanowires functionalized with antibodies specific for influenza A virus particles with a microfluidic sampling system. The nanowire sensing system was able to detect influenza A at the single-virus level, demonstrating that single virus/nanowire recognition events can be detected by measuring real-time changes in nanowire conductivity. In addition to silicon nanowires, metallic multi-striped nanowires have also shown great promise as potential platforms for multiplex immunoassays. These nanowires are built through submicrometer layering of different metals, e.g., gold, silver and nickel, by electrodeposition within a porous alumina template. Due to the permutations in which the metals can be deposited, a large number of unique yet easily identifiable encoded nanowires can be fabricated. The multi-striped nanowires, when coated with target-specific antibodies, were reported to efficiently and accurately allow multiplex detection of Bacillus globiggi spores, MS2 bacteriophage and ovalbumin protein. The sensitivity of detection for B. globiggi spores, MS2 bacteriophage and ova protein was estimated to be 1ˆó10⁵ cfu/ml, 1ˆó10⁵ pfu/ml and 5 ng/ml, respectively, and is comparable with results obtained using microarrays.

Nanowires are set apart from other available nanobiotechnologies due to several key features such as direct, label-free, real-time electrical signal transduction, as well as ultrahigh sensitivity, exquisite selectivity and the potential for integration of addressable arrays. However, an intrinsic limitation of nanowires is that the detection sensitivity depends on solution ionic strength. Consequently, for samples with a high ionic strength, diagnostics will require a desalting step before analysis to achieve the highest sensitivity. Furthermore, a practical constraint might be that the synthesis and fabrication of nanowire biosensor devices require some technologies that are not common to most laboratories.

Nucleic acid hybridization techniques
The easiest way of detecting specific nucleic acid sequences is through direct hybridization of a probe to microbial nucleic acid extracts. These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). The probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specific to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g., ease of synthesis and stability), most studies have employed DNA probes. The probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots).

According to their length, DNA probes can be grouped as either polynucleotide probes (more than 50 nucleotides) or as oligonucleotide probes (less than 20 nucleotides). The latter are more frequently used since they are short enough to allow for single mismatch discrimination of target nucleic acids and large quantities of oligonucleotides can be rapidly and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5' end of the oligonucleotide. The principle conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used. For probes to be effective in nucleic acid hybridization, they must be specific and selective. This requires that sequence data be available. Although such data is available for some waterborne pathogens, the data are not comprehensive for all pathogens of concern. However, the use of bacterial 16S rRNA sequences to develop determinative hybridization probes is now well established. The occurrence of 16S rRNA molecules in high copy numbers of usually more than 1000 in any living cell, as well as the fact that 16S rRNA sequences have been determined for a large fraction of bacterial species, currently confers on these genes a high informative value at phylogenetic level. Comparative analyses of rRNA sequences have indicated that the rRNA molecules comprise highly conserved sequence domains interspersed with more variable regions. Consequently, so-called signature sequence motifs on various taxonomic levels, from domains to subspecies, can be identified. Whereas species-specific probes complement the most variable regions, more general probes target more conserved regions of the molecule. Although several oligonucleotide probes are commercially available, new specific probes may also be designed. The design of such 16S rRNA-targeted probes is greatly facilitated by computer-aided sequence comparisons of large rRNA sequence databases. The principle steps involved in the design of probes are: the alignment of rRNA gene sequences; the identification of sequence idiosyncrasies; the synthesis and labelling of complementary nucleic acid probes; and the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains.

Nucleic acid hybridization can be performed in a variety of formats. The procedure typically entails the extraction of whole-cell DNA or RNA from the sample that is subsequently fixed to a nylon or nitrocellulose membrane. Alternatively, bacterial colonies can be replica-plated from agar plates to membranes and their nucleic acids exposed following lysis for subsequent hybridization. Whereas the former direct approach is usually inadequate due to high detection limits and large sample volumes that are impractical for most hybridization protocols without further pathogen concentration, the latter approach is time-consuming and cannot be applied to nonculturable organisms. Consequently, the use of whole-cell fluorescent in situ hybridization (FISH) as a method for the direct detection of bacteria in water samples has increased in popularity. The method typically combines membrane filtration, resuscitation of the bacteria on membranes and hybridization with rRNA-targeted probes. During FISH, the morphology of the cells in the sample has to be stabilized in order to maintain the morphological integrity of the cells under harsh hybridization conditions. The cell walls and membranes have to be permeabilized to allow free penetration of fluorescent oligonucleotides to the intracellular rRNA. This can be achieved with fixatives such as aldehydes and/or alcohols. The membranes are hybridized in hybridization solution containing a fluorescently-labelled oligonucleotide. Following incubation at the hybridization temperature for one to several hours, to allow the probe to bind to complementary rRNA sequences, washing steps are used to remove unbound or the non-specifically bound fluorescent probe, and the hybridized cells are then detected by epifluorescence microscopy. Probes will only bind correctly under defined hybridization conditions and the optimization of hybridization and washing conditions is as important as the probe design. Several probes labelled with spectrally different fluorochromes can be simultaneously used on one sample, while counterstaining of the fixed cells with DAPI (4',6-diamidino-2-phenylindole) allows total counts to be made. Alternatively, depending on the concentration of targeted cells in the sample and to increase resolution, FISH hybridization can be performed in suspension and detection can be performed by means of flow cytometry, which enables quantification of the fluorescence intensities for each target-probe hybrid.

Although FISH is currently considered a highly specific cellular detection method and is relatively easy to perform, the procedure may have some limitations when applied to the detection of nutrient-starved bacterial cells disseminated in drinking waters. Generally, probes give a strong signal only if cells are metabolically active and contain large numbers of ribosomes and target rRNA. Conversely, low cellular ribosome contents may result in weak fluorescent hybridization signals. Another area of concern relates to the viability of the detected cells. Although it has been reported that the rRNA content of bacterial cells can be directly correlated with the growth rate, some reports have indicated that a small number of rRNA molecules can remain for a relatively long period after the loss of culturability, thus resulting in false positives. One possible way to overcome this issue is to couple direct viable count techniques with FISH detection.

Generally, microorganisms, in particular prokaryotes often lack morphological and behavioral characters amenable to phylogenetic analysis. Such a lack of information in these areas makes gene sequence information the most prevalent source of data for phylogenetic analysis in pre-genomic era. Molecular phylogenetics based on single genes, in particular the small-subunit rRNA (SSU rRNA), has laid the foundation for a modern classification system, conceptually represented by the 'universal tree of life'. However, phylogenetic trees based on single genes or gene families may show conflict results due to a variety of problem, such as mutational saturation of the single genes and horizontal gene transfer. Consequently, although SSU rRNA gene sequence continue to be considered as molecular criteria for species delineation, it is anticipated that much additional taxonomic information can be extracted from complete genome sequences.

Now that large-scale genome-sequencing projects are sampling many organismal lineages, it is becoming possible to compare large data sets of DNA or protein sequences to study speciation and evolution. The steady increase in the number of completely sequenced prokaryotic genomes has created a boom for bioinformatics. With more than 700 prokaryotic genomes completely sequenced, there has been an increasing interest in the use of various characters in whole genomes for prokaryotic genomes studies. This is giving birth to a brand new field of research - phylogenomics. Phylogenomics use entire genomes to infer a species tree and has become the de facto standard for reconstructing reliable phylogenies.

One major branch of phylogenomics involves the use of these data to reconstruct the evolutionary history of organisms. Access to large amount of genomic data could potentially alleviate problems associated with single-gene based phylogenetics. This is because a large number of characters can now be used for phylogenetic analysis to avoid biases. With this increase, the emphasis of phylogenetic inference is shifting from the search for informative characters to the development of better reconstruction methods using genomic data. Existing models used in tree-building algorithms only partially take into account molecular evolutionary processes, and phylogenomic inference will benefit from an increased understanding of these mechanisms. Interestingly, phylogenomics is also providing the opportunity to use new 'morphological-like' characters that are based on genome structure, such as rare genomic changes (RGCs). The integration of genomics data into the phylogenetics is still at an early stage. Given the breadth of organismal diversity, the gene-scale era of phylogenetics is still an invaluable asset to the pursuit of the Tree of Life. Comparative genomics, with its ability and potential to vastly increase both the amount and type of molecular data available for a small but critical fraction of biodiversity, is bound to play an increasingly important role in efforts to assemble a robust picture of the Tree of Life.

BPhyOG A database for overlapping genes in prokaryotic genomes
BPhyOG (Bacterial Phylogeny based on Overlapping Genes) is an online interactive server for reconstructing completely sequenced bacterial genomes on the basis of their shared overlapping genes. It provides two tree-reconstruction methods: Neighbor Joining (NJ) and Unweighted Pair-Group Method using Arithmetic averages (UPGMA). Users can apply the desired method to generate phylogenetic trees, which are based on an evolutionary distance matrix for the selected genomes. The distance between two genomes is defined by the normalized number of their shared OG pairs. BPhyOG also allows users to browse the OGs that were used to infer the phylogenetic relationships. It provides detailed annotation for each OG pair and the features of the component genes through hyperlinks. Users can also retrieve each of the homologous OG pairs that have been determined among 177 genomes. BPhyOG is useful tool for analyzing the tree of life and overlapping genes from a genomic standpoint. It currently includes 177 completely sequenced bacterial genomes containing 79,855 OG pairs, with their annotation and homologous OG pairs comprehensively integrated. The reliability of phylogenies and completeness of annotations make BPhyOG a comprehensive and powerful web server for genomic and genetic studies.

As a consequence of the speed, specificity and low cost of the PCR, the procedure has become one of the most widely used assays for direct detection of low levels of pathogenic microbes in environmental samples. The PCR assay can be used to selectively amplify, to detectable levels, nucleic acid sequences associated with pathogens that might be present in low numbers in water samples. PCR is a process in which target DNA, synthetic oligonucleotide primers, a thermostable DNA polymerase and the DNA subunits are combined in a microcentrifuge tube and subjected to the temperature changes needed for the DNA duplication to occur. During the PCR process, different temperatures are used to facilitate DNA denaturation, annealing of the oligonucleotide primers to the target DNA and extension of the primers across the target sequence. These cycles are repeated many times, thus resulting in increasingly greater quantities of target sequence. Under ideal conditions, the PCR can generate millions of copies of a single DNA molecule in just 20 to 30 repetitions of the temperature cycle, with each cycle requiring only a few minutes. The PCR-amplified products can be detected by means of techniques such as electrophoresis on agarose gels and after staining of the amplification products by a fluorochrome dye or by hybridization with a labelled probe.

There are several essential steps in the development and application of PCR for successful detection of pathogens in water samples. The key steps include: identification and selection of oligonucleotide primers for target genomic sequences; testing of selected primers for sensitivity, specificity and selectivity; purification and concentration of the pathogens in environmental sample concentrates to enable efficient and reliable enzymatic amplification of low numbers of target genomic sequences; and testing of the methods for their applicability to natural pathogenic strains and actual field samples. By using PCR, a selected gene sequence specific to a group of organisms or a single species can be selectively amplified. Various primers have thus been described to amplify fragments of rRNA operons in order to detect specific organisms or groups of organisms in environmental samples. As in the case of designing oligonucleotide probes for hybridization purposes, selection of oligonucleotide primers for target pathogens requires that sequence data be available. Of particular importance is the type and function of the nucleic acid target; its length and location; and the extent to which its sequence is related to that of other, nontarget but genetically related microbes. It is essential to select oligonucleotides having an appropriate length (20 to 30 nucleotides); desired sequence composition for specificity and selectivity; and appropriate melting and annealing temperatures to prevent the formation of undesirable secondary structures, primer-dimers and other artifacts that would interfere with successful PCR.

There exist many variations of the basic PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. Nested PCR involves two consecutive rounds of PCR amplification. The first round of amplification is performed with group- or organism-specific primers, whilst the second round of amplification uses the initially amplified product as the template for another round of annealing and extension with different primers. The use of nested primers provides an additional level of specificity since the second round of PCR amplification can only be performed if the correct sequence (complementary to the nested primers) was amplified during the first round. Alternatively, nucleic acid hybridization can be performed using highly specific oligoprobes that would hybridize only with amplicons from a single pathogen type or strain. However, nested PCR permits a more rapid detection (6 to 8 h) compared to confirmation of the correct sequence amplification by probe hybridization (few days). Another variation of the PCR technique, namely multiplex PCR, allows the simultaneous detection of more than one target organism in a single PCR using multiple pairs of primers designed to be specific for different target organisms. However, multiplex PCR may not perform well with all primer blends as the composition and length of primer oligonucleotide, as well as the size of the amplified fragments, may influence each PCR amplification. Since the PCR method cannot be used directly for the amplification of an RNA target sequence, a complementary DNA copy (cDNA) thereof must first be synthesized. This reaction is catalyzed by the enzyme, reverse transcriptase (RT), which is able to synthesize DNA from the RNA template in the presence of specific primers and DNA subunits. The single-stranded cDNA is a suitable target for PCR amplification by making use of the same or a different set of primers. RT-PCR has emerged as a sensitive and specific approach for the detection of enteric viruses containing RNA genomes.

Although the basic PCR method is both specific and sensitive, such standard PCR reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used. In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction. Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the PCR reaction mixture. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from the complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product after which a deletion is introduced to give a differently sized PCR product.

An alternative PCR assay for the direct enumeration of targeted cells was reported by Tani et al., who modified the standard PCR protocol so that nucleic acid sequences can be amplified in situ. This new PCR method was successfully applied to the direct enumeration of E. coli from a freshwater sample. With proper fixation and permeabilization conditions, the oligonucleotide primers and other reaction components are able to diffuse into the cells, and, upon thermal cycling, amplify a specific target sequence present in the cell. PCR products were labelled by digoxigenin during the amplification process and anti-digoxigenin antibodies conjugated with fluorescent dye were used for detection by epifluorescence microscopy. This approach allows direct visualization of the fluorescent amplification products at a single-cell level and consequently, direct enumeration of cells. Even though in situ PCR seems promising, it has not been used for routine detection and enumeration of microorganisms in water, as the results showed a weak fluorescence intensity signal of targeted cells.

Another promising approach for quantifying the number of cells is real-time quantitative PCR, which consists of monitoring fluorescently-labelled PCR products as they are being amplified. The fluorescent signal can be generated by using an intercalating fluorescent dye (e.g., SYBR Green I or SYBR Gold) or a probe system (e.g., TaqMan). The use of intercalating dyes is the simplest and least costly approach and involves adding the fluorescent dye directly to the PCR. These dyes undergo a conformational change to become a more efficient fluorophore on binding to double stranded DNA (dsDNA). Although SYBR Green I-based assays are very sensitive, the primer's specificity for the target is crucial as any dsDNA is detected, including any primer artifacts, which can lead to false positive results. Moreover, multiplex reactions are impractical since the dye binds to all dsDNA. The TaqMan approach depends on oligonucleotide probes complementary to a sequence located between the two primers used for PCR amplification. At one end of the probe a fluorescent reporter dye is conjugated, whilst at the other terminus there is a quencher that may be another fluorophore (also called dark-quencher). In effect the structure possesses two dyes in close proximity and in this configuration the fluorescence of one (the reporter) is quenched by the other through FRET (fluorescence resonance energy transfer). During the extension step of PCR the DNA polymerase degrades the bound TaqMan probe, using its inherent 5'-3' exonucleolytic activity, and thus results in the separation of reporter from quencher and an increase in fluorescence emission of the reporter molecule. Although this approach is less prone to false positive results compared to the use of intercalating fluorescent dyes, it is more expensive due to the requirement of the probe molecule. However, by choosing the fluorophores astutely it is possible to perform multiplex PCR. During real-time PCR, irrespective of the approach used, the accumulation of amplified product is measured automatically at each PCR cycle. The amount of target sequence is deduced from the number of PCR cycles (threshold cycle or Ct) required to cross a fixed point above a baseline, using a standard curve as reference. External quantification standards for the construction of standard curves of Ct versus copy number usually consist of the target sequence cloned into a plasmid or DNA extracted from cultured cells where the concentration or copy number of the target can be determined accurately.

Although there are numerous advantages associated with PCR as detection tool, standard PCR cannot, however, be used to detect the infectious state of an organism - only the presence or absence of pathogen-specific DNA or RNA. Yet, this viability concept is fundamental for interpreting the result in terms of public health when dealing with water samples. The PCR technique must consequently be associated with a viability test. To overcome this limitation, an indirect approach has been developed for assessing the viability of PCR-detected bacteria from water samples. This method is based on the analysis of each sample before and after a culture step in a nonselective medium: an increase in the PCR-amplified product after cultivation indicates the occurrence of bacterial multiplication and thus demonstrates the viability of the detected bacteria. Recently, a PCR-based approach to limit detection to intact (viable) cells with an active metabolism has been reported. The approach is based on the use of ethidium monoazide (EMA), which is suggested to enter only membrane-compromised cells (considered "dead"). Once inside membrane-compromised cells, EMA intercalates into the DNA and is covalently bound to the DNA after exposure of the treated samples to bright visible light, whilst the unbound EMA, which remains free in solution, is simultaneously inactivated by reacting with water molecules. The EMA treatment is followed by extraction of genomic DNA and analysis by PCR. The result of treatment is that only unmodified DNA from intact cells whose DNA was not cross-linked with EMA can be amplified, whereas PCR amplification of modified DNA from membrane-compromised cells is efficiently suppressed. Treatment was thus suggested to lead to the exclusion of cells with damaged membranes from analysis.

Despite its advantages, accurate characterization or identification of microbes by PCR is influenced by the same bias and variations that are inherent in many nucleic acid techniques. The main concerns are biased nucleic acid extraction (e.g., efficiency of extraction or cell lysis if using whole-cell methods), degradation of nucleic acids by nucleases and primer reactivity (i.e. sensitivity, specificity and accessibility). Additionally, a frequently encountered limitation inherent to PCR analysis of environmental samples is the inhibition of the enzymatic reaction. Whereas humic substances are known to inhibit the DNA polymerase enzyme, colloidal matter has a high affinity for DNA. The presence of these elements in a water sample can therefore considerably decrease the amplification yield of PCR applied to the detection of greatly diluted bacteria. Consequently, for PCR or RT-PCR, the extracted target nucleic acid is purified by protocols utilizing, for example, Sephadex, Chelex or CTAB.

PCR was destined to be a quantitative technique. By both theory and practice, a well optimized PCR doubles the amount of product each cycle for many cycles. Early attempts to harness the quantifying power of PCR were limited by dependence on end-point analysis of the products generated, either by removal of an aliquot of the reaction at predetermined cycle numbers (PCR cycle titration) or serial dilution PCR (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Additional attempts were made to measure PCR products in the log phase of the reaction or include a competitive internal control in the reaction. These methods were time-consuming and labor intensive, often using agarose gels to quantify the amount of PCR product and from this determine an initial template concentration (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Real-time PCR greatly simplified quantification. By monitoring fluorescence once each cycle, fluorescence as a surrogate of PCR product amount can be plotted against cycle number. No longer is there a need to physically sample a reaction at multiple cycles or guess when PCR is exponential. By acquiring data at all cycles, exponential data can be selected in retrospect. The exponential region is identified by plotting fluorescence on a log plot and the earliest cycle "significantly above background" chosen to correlate with the initial template amount. Such quantification cycles (Cqs) are usually determined by either a fluorescence threshold or by the maximum second derivative. In either case, these fractional cycle numbers are inversely related to the log of the initial template concentration. Technical aspects of qPCR and performance guidelines have recently been published (For details see: Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Terms such as "rapid" or "fast" are relative and vague. A 1 hour PCR is fast compared to 4 hours, but slow compared to 10 min. Furthermore, faster PCR is possible if you start with a higher template concentration or use fewer cycles. It is better to define the time required for each cycle and rapid-cycle PCR has been defined as 30 cycles in 10-30 min (See: Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization) so that each cycle is 20-60 s each. The actual time of each cycle is longer than the sum of the times programmed for denaturation, annealing and extension. Indeed, during rapid PCR the temperature is usually changing. This challenges the "equilibrium paradigm" of PCR, where 3 reactions (denaturation, annealing and extension) occur at 3 temperatures over 3 time periods each cycle (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Although intuitive, the equilibrium paradigm does not fit well with physical reality. Instantaneous temperature changes do not occur and reactions occur over a range of temperatures at different rates. More accurate is a kinetic paradigm for PCR where reaction rates and the temperature are always changing. Under the equilibrium paradigm, a cycle is defined by three temperatures each held for a time period, whereas the kinetic paradigm requires transition rates and target temperatures(Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Paradigms are not right or wrong and should be judged by their usefulness. The equilibrium paradigm is simple to understand and lends itself well to the engineering mindset. The kinetic paradigm is more relevant to biochemistry, rapid PCR and melting curve analysis. Although most commercial instruments still follow equilibrium protocols, rapid protocols are a nice match for microsystems, where small volumes and rapid PCR are natural (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Rapid-cycle PCR is used in real-time instruments such as the Roche carousel LightCycler and Cepheid's SmartCycler. Other companies now promote "Fast" protocols on more conventional thermal cyclers. Few instruments based on microtiter plates and heat blocks can approach rapid-cycling speeds and rapid PCR does not require special reagents.

The first two commercial real-time PCR platforms were the ABI 7700 and the LightCycler. The LightCycler was initially developed through a small business NIH grant. The prototype, constructed at the University of Utah, integrated rapid temperature cycling with fluorescent monitoring adapted from a flow cytometer. Idaho Technology converted the prototype to a 24-sample instrument with a small footprint and simplified optics for commercial sale. In 1997, the system was licensed to Boehringer Mannheim which was subsequently acquired by Roche that same year. A 32-sample LightCycler was released by Roche in 1998 integrating rapid-cycling, SYBR Green I, dual hybridization probes and melting curve analysis (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The ABI 7700 was a large, plate-based 96-well instrument focused on hydrolysis probes.The 7700 used a 488 nm laser and fiber optics, in contrast to the light emitting diodes and epifluorescence optics of the LightCycler. Today there are many product offerings in the arena of real-time instrumentation. Competition has driven down the costs of instruments and reagents.

Real-time PCR not only automates both amplification and detection, but integrates them so that they occur concurrently. Time, temperature and fluorescence are monitored during PCR in real-time instruments. The earliest report of continuous monitoring of PCR and acquiring fluorescence at each cycle utilizing ethidium bromide, a double-stranded DNA (dsDNA) specific dye. This allowed for a truly homogenous or "closed-tube" assay in which product amplification was combined with detection. The most important application of real-time PCR is quantification of the initial template, known as quantitative PCR or qPCR (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

PCR specificity under ideal conditions is extraordinary. However, the genomes are large and primers may bind not only to their intended target but also to other areas of the genome. Furthermore, the primers in PCR are at high concentrations, so even minor self or cross complementation may initiate primer dimers. These side reactions can create so-called "non-specific" products other than the desired product. A number of methods have been developed to prevent primer extension at low (room) temperatures where polymerase activity, although greatly reduced, is still capable of extending primers (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The first "hot-start" techniques relied on adding an essential reaction component (such as the polymerase) only after the reaction had reached high temperatures to favor specific primer annealing. This required opening the reaction container and increased the possibility of PCR contamination. To circumvent this problem, waxes and greases were used to physically partition reagents with a barrier that would melt at high temperature, mixing the essential reagent(s) with the other reaction components (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Instead of physical separation, polymerase activity can be inhibited at room temperature. For example, monoclonal antibodies against the active site of the polymerase can inhibit the enzyme until they denature at high temperature. Alternatively, the polymerase active site can be chemically modified by heat-labile covalent modifications that break down and activate the enzyme at high temperature. Instead of inactivating the polymerase, oligonucleotide primers or dNTPs can be modified at their 3'-end with similar heat-labile linkages (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Many different reagents are now available to augment PCR specificity, but they are usually only necessary when the template copy number is low. Nevertheless, such reagents are an easy way to increase the robustness of PCR, sometimes making optimization unnecessary. If a hot start method is required, the best method depends on the circumstances. For example, an antibody-mediated hot-start is more useful in rapid PCR because chemically modified polymerases typically require 15-30 min for activation, longer than an entire rapid-cycle PCR protocol.

The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. Early evolution of the PCR process included adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.

With 3 billion bases in the human genome, it is not easy to find and analyze the small sequence regions that confirm a genetic disorder, identify an oncogenic change or detect microbial infection. The polymerase chain reaction (PCR) provides this focus. Since its development 25 years ago, it has become the most important tool for working with nucleic acids in molecular biology and clinical diagnostics. It deserves such central recognition because of its simplicity.

Before PCR, molecular methods were multi-stepped, laborious and time consuming. To amplify DNA, it had to be cloned into plasmids, the plasmids inserted into bacteria, the bacteria grown in culture, the bacteria harvested, the plasmids isolated from the bacteria, and the DNA inserts separated from the plasmid DNA. Southern blotting required multiple steps of restriction enzyme digestion, electrophoresis, blotting onto membranes, hybridization with radioactively-labeled oligonucleotide probes and development on X-ray film. These early techniques required large amounts of DNA and strong technical expertise for consistent results.

PCR greatly reduced the number of steps required to generate appreciable quantities of DNA necessary for many applications. The acceptance of PCR in the scientific community was relatively swift, with an independent research group using the technique within a year. PCR has revolutionized molecular biology and clinical diagnostics. (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization)

PCR is simple and elegant. It is remarkably robust and tolerates the addition of many diverse reagents such as electrophoresis dyes (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Vaccine Design: Innovative Approaches and Novel Strategies Edited by: Rino Rappuoli and Fabio Bagnoli ISBN: 978-1-904455-74-5 Publisher: Caister Academic Press Publication Date: February 2011 Cover: hardback

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PCR Troubleshooting and Optimization: The Essential Guide Edited by: Suzanne Kennedy and Nick Oswald ISBN: 978-1-904455-72-1 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback

read more ...]]> ConferencesMolecular Biology ConferenceMolecular Biology ConferencesFri, 22 Oct 2010 07:32:44 -0400http://www.caister.com/molecular-biology-blog/files/conference-update-october-2010.html#unique-entry-id-77http://www.caister.com/molecular-biology-blog/files/conference-update-october-2010.html#unique-entry-id-77Waltham, MA, USA Further information
Meeting on RNA interference: Biochemistry to Drugs and Therapeutics
Suggested reading: RNA Interference and Viruses: Current Innovations and Future Trends

April 4 - 5, 2011 First International Single (Molecule) Cell Biology and Real-Time PCR Meeting 2011
Waltham, MA, USA Further information
Meeting‚ on Single Molecule Detection to Amplification and Molecular Imaging
Suggested reading: Real-Time PCR books

July 11 - 12, 2011 Fourth International Epigenomics, Sequencing and SNiPs Meeting 2011
Boston, MA, USA Further information
Meeting on Chromatin Methylation to Disease Biology and Theranostics
Suggested reading: Epigenetics]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:47:35 -0400http://www.caister.com/molecular-biology-blog/files/chromatin-structure.html#unique-entry-id-76http://www.caister.com/molecular-biology-blog/files/chromatin-structure.html#unique-entry-id-76Cambridge, UK Further information
A joint Biochemical Society / Wellcome Trust conference. The meeting will present advances in our understanding of chromatin structure and organization and the impact of these findings on our understanding of biological function. The meeting will bring together experts in the field to present recent advances in structural models for chromatin and the role of accessory factors that regulate its organization. Topics to be covered will include the structure of the nucleosome core particle and the consequences resulting from histone modification and inclusion of histone variants, the analysis of nucleosome positioning in vivo and DNA sequence periodicities that influence location. Wider perspectives to be addressed include the relationship of chromatin and DNA repair, the role of high mobility group proteins and linker histones in chromatin organisation and function, and models for hierarchical assembly of chromatin. The meeting will mark the retirement of Professor Jean Thomas, who has made major contributions to the field.
Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:46:53 -0400http://www.caister.com/molecular-biology-blog/files/glutathione.html#unique-entry-id-75http://www.caister.com/molecular-biology-blog/files/glutathione.html#unique-entry-id-75Sant Feliu de Guixols, Spain Further information
The conference format includes lectures by invited high-level speakers, short talks by young and early stage researchers, poster sessions, and a Forward look discussion session. We encourage off the record presentations of previously unpublished scientific results. Young scientists are encouraged to apply for a meeting and travel grant. This conference is organised by the European Science Foundation (ESF), in partnership with EMBO.
Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:45:38 -0400http://www.caister.com/molecular-biology-blog/files/biotechnology-conference.html#unique-entry-id-74http://www.caister.com/molecular-biology-blog/files/biotechnology-conference.html#unique-entry-id-74Manila, Philippines Further information
The Philippine Society for Microbiology Inc (PSM) convenes the Asia-Pacific Biotechnology Congress every five years to promote multi-country sharing of breakthroughs and collaborative research on microbial biotechnology. The conference will be hosted alongside the alongside its 40th Annual Convention and Scientific Meeting of the PSM. The event, with the theme: Microbiology and Biotechnology: Rising to the Challenges of the Times, will be a venue for the convergence of microbial technology researchers and experts from the Philippines and the Asia Pacific region.
Suggested reading: Biotechnology Books]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:44:50 -0400http://www.caister.com/molecular-biology-blog/files/plant-gene-discovery-technologies.html#unique-entry-id-73http://www.caister.com/molecular-biology-blog/files/plant-gene-discovery-technologies.html#unique-entry-id-73Vienna, Austria Further information
Reverse Genetics and Mutants Technologies; Gene Discovery by TILLING; DNA Sequencing Technologies; Protein Sequencing and Protein Discovery; Microarrays; Protein Microarrays; Gene Discovery using OMICS; Bioinformatics; Evaluation and Application of Sequencing; System Biology and Gene Network Discovery. Amongst the invited speakers are internationally known names such as G.W. Haughn, J.-K. Zhu, B.J. Till, S.B. Gelvin, P. Ronald, L.O. Lomas, C.J. Mann, R.L. Last, K. Shinozaki, B.C. Meyers, C. N. Stewart, Jr., J.-F. Gibrat, K. Mitchelson, M.Matsui, V. Bafna, B. Ulker and others.
Suggested reading: Recent Advances in Plant Virology]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:43:53 -0400http://www.caister.com/molecular-biology-blog/files/plant-transformation.html#unique-entry-id-72http://www.caister.com/molecular-biology-blog/files/plant-transformation.html#unique-entry-id-72Vienna, Austria Further information
Agrobacterium Mediated Plant Transformation; Particle Bombardment and Other Transformation Methods; Explants Used for Plant Transformation; Transformation of Important Crops; Plant Transformation Tools: Genes, Vectors, Promoters etc; Selectable and Screenable Markers; Molecular Analysis of Transgenic Events and Transformmants; Expression of Transgenes in Transgenic Plants (Integration, Stability); Marker Excision and Marker Free Transtechnologies; Plastid Transformation and Biotechnology; Transgenic Plants as Bio Factories; Transgenic Plants and Public; Intellectual Property in Plant Transformation; Emerging Plant Trans-Technologies. Amongst the invited speakers are internationally known names such as M. Van Montagu, C.N. Stewart, Jr., V. Citovsky, S.B. Gelvin, H.D. Jones, H. Kobayashi, D.W. Ow, R. Bock, M. Boutry, L.-Y. Lee, A. Trewavas, G. Lomonossoff, G. Corrado, S.-S. Kwak, G. Angenon, H. Daniell and others.
Suggested reading: Recent Advances in Plant Virology]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:42:47 -0400http://www.caister.com/molecular-biology-blog/files/gene-targeting.html#unique-entry-id-71http://www.caister.com/molecular-biology-blog/files/gene-targeting.html#unique-entry-id-71Vienna, Austria Further information
Topics covered include: Homologous Recombination and Repair; Gene Targeting in Multicellular Eucaryotes; Gene Targeting in Mammals; Gene Targeting in Plants; Gene Targeting in Human; Zink Finger Nucleases; Gene Targeting by Oligonucleotides; Gene Targeting and Cancer. Amongst the invited speakers are internationally known names such as Sir A. Klug (Nobel Prize Winner), Y.S. Rong, A. Levi, P. Baumann, T. Tzfira, M.H. Porteus, D. Voytas, D. Carroll, D.W. Russell, H. Te Riele, R. H. Friedel, J.S. Owen, N. Adachi, A.M. Carr, Y. Hong and others.
Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceMolecular Biology ConferencesTue, 12 Oct 2010 05:41:34 -0400http://www.caister.com/molecular-biology-blog/files/cell-cycle-regulators.html#unique-entry-id-70http://www.caister.com/molecular-biology-blog/files/cell-cycle-regulators.html#unique-entry-id-70Vienna, Austria Further information
Topics covered include: Cell Cycle Regulation; Mitosis, Antimitotic Drugs; Mitotic Spindle, Microtubule Inhibitors/Regulators; Anaphase and Anaphase Promoting Complex Inhibitors/Regulators; Histone Deacetylase Inhibitors/Regulators; Cell Cycle Inhibitors and Cancer; Kinase Inhibitors and Cancer; Methodologies/Technologies. Amongst the invited speakers are internationally known names such as A. Giordano, C. L. Rieder, J. M. van Deursen, S. Linardoloulous, U. Surana, H.M. Coley, Y. Pommier, H. Bastians, G. J. Gorbsky, W. Tao, A. Gilmartin, T.D. Halazonetis, H. Hirai, R.C. Jackson, P.T. Daniel and others.
Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceMolecular Biology ConferencesWed, 29 Sep 2010 09:59:08 -0400http://www.caister.com/molecular-biology-blog/files/hh-gli-signaling.html#unique-entry-id-69http://www.caister.com/molecular-biology-blog/files/hh-gli-signaling.html#unique-entry-id-69Kolymbari, Crete, Greece Further information
The goal is to bring together a diverse group of scientists studying various aspects of Hegdehog-Gli signalling. Highlights on regeneration, healing, stem cells and cancer in humans and multiple systems including mice and flies will be presented. This meeting is part of an ITN EU-funded network.
Suggested reading: Molecular Biology Books

]]> Biochemical Society ConferencesWed, 29 Sep 2010 09:57:26 -0400http://www.caister.com/molecular-biology-blog/files/biochem-soc-conferences.html#unique-entry-id-68http://www.caister.com/molecular-biology-blog/files/biochem-soc-conferences.html#unique-entry-id-68Durham, UK Further information
Biochemical Society Conference. Significant new advances have been made recently in the understanding of inflammatory bowel disease (IBD) pathology at the molecular biology level. This Biochemical Society Focused Meeting has been planned to cover some of the major issues currently being considered. Topics that have been selected relate to; the identification of IBD susceptibility genes and disease markers; innate and adaptive immune systems in IBD pathogenesis; the function of the epithelial protective barrier; interactions of the enteric bacterial flora with the human host leading to normal and pathological regulation of the immune system and links with nutrition and probiotics and finally the value of mouse models of ulcerative colitis in examining molecular disease mechanisms.
Suggested reading: Molecular Biology Books   Bifidobacteria: Genomics and Molecular Aspects   Lactobacillus: From Genomics to Probiotics

April 18 - 19, 2011 Analysis of free radicals, radical modifications and redox signalling
Birmingham, UK Further information
Biochemical Society Conference. Redox signalling via reversible modifications of protein thiols and bioactivity of oxidized lipids are topical fields that underpin understanding of physiological and disease processes. This meeting will focus on state-of-the-art methodology for measuring cellular levels of free radicals and analysing biomolecule oxidation, including discussion of the applications and limitations.
Suggested reading: Molecular Biology Books

January 10 - 12, 2012 Frontiers in Biological Catalysis
Cambridge, UK Further information
Annual Symposium of the Biochemical Society. The symposium places great emphasis on the role of catalysis in key biological processes, ranging from signalling, apoptosis, respiration and photosynthesis, to antibiotic synthesis and related secondary metabolism. The meeting will seek to emphasise the detailed/unique information emerging from reductionist approaches, and how to integrate with high-throughput approaches characteristic of the systems biology era. Reductionist approaches are particularly powerful when applied in a multidisciplinary setting, and there is the need to transcend the inherent limits of individual disciplines (e.g. crystallography, molecular modelling) to provide state-of-the-art dynamic insight into enzyme function.
Suggested reading: Molecular Biology Books

]]> Book ReviewTue, 28 Sep 2010 12:17:12 -0400http://www.caister.com/molecular-biology-blog/files/book-review-phylogeny.html#unique-entry-id-67http://www.caister.com/molecular-biology-blog/files/book-review-phylogeny.html#unique-entry-id-67Molecular Phylogeny of Microorganisms:

"Molecular phylogeny, the analysis of gene or protein sequences to unravel the relatedness among microorganisms, plays an important role in microbial taxonomy. One of the most exciting developments in this respect was the discovery of the two domains, Bacteria and Archaea, by Carl Woese in the 1970th. The present book, Molecular Phylogeny of Microorganisms, edited by Aharon Ohren and R. Thane Papke, describes very nicely the different approaches to apply molecular phylogeny, encountering the difficulties with the present phylogenomic species concept. This book ... addresses the most interesting issues in relation to molecular phylogeny ... Anyone, who is interested in microbial phylogeny, surely will enjoy reading this book. The hardcover, format and descriptions of the book make it a pleasure to read." from Mareike Viebahn (Centocor BV, Leiden, The Netherlands) writing in Curr. Issues Mol. Biol. read more ...

Molecular Phylogeny of Microorganisms Edited by: Aharon Oren and R. Thane Papke ISBN: 978-1-904455-67-7 Publisher: Caister Academic Press Publication Date: July 2010 Cover: hardback "a pleasure to read" (Curr. Issues Mol. Biol.)

]]> BiotechnologyThu, 09 Sep 2010 06:11:23 -0400http://www.caister.com/molecular-biology-blog/files/clavulanic-acid.html#unique-entry-id-66http://www.caister.com/molecular-biology-blog/files/clavulanic-acid.html#unique-entry-id-66Clavulanic Acid and Clavams Biosynthesis and Regulation
from Paloma Liras, Irene Santamarta and Rosario Pérez-Redondo writing in Streptomyces: Molecular Biology and Biotechnology:

The (3R,5R) clavulanic acid and (3S,5S) clavam molecules share a structure formed by a four member β-lactam and a five member oxaxolidine ring and have several initial common steps in their biosynthesis pathways. The precursors of the molecules are glyceraldehyde-3-phosphate and arginine, condensed by the carboxyethylarginine synthetase (CeaS). The next steps in the pathway occur by the subsequent action of the β-lactam synthase (Bls) forming the β-lactam ring, a proclavaminic acid guanidine hydrolase (PAH) and the clavaminate synthase (Csa2), which forms the two rings clavam structure of clavaminic acid. Modifications of this compound result in late step intermediates for clavulanic acid biosynthesis, N-glycylclavaminic acid or clavaldehyde, and in the clavams structures. In addition to the clavulanic acid gene cluster, two additional clusters containing paralogous genes for clavulanic acid biosynthesis and clavam biosynthesis have been located in S. clavuligerus. Biochemical characterization of the clavam non producer mutants will clarify the biosynthetic pathway of these compounds.

Further reading: Streptomyces: Molecular Biology and Biotechnology]]> BiotechnologyThu, 09 Sep 2010 06:10:04 -0400http://www.caister.com/molecular-biology-blog/files/actinomycetes-gene-clusters.html#unique-entry-id-65http://www.caister.com/molecular-biology-blog/files/actinomycetes-gene-clusters.html#unique-entry-id-65Gene Clusters for Bioactive Natural Products in Actinomycetes and their Use in Combinatorial Biosynthesis
from Carlos Olano, Carmen Méndez and José A. Salas writing in Streptomyces: Molecular Biology and Biotechnology:

During the last twenty five years the isolation and characterization of gene clusters involved in the biosynthesis of actinomycete secondary metabolites has permitted the elucidation of the biochemical steps involved in the production of different structural classes of bioactive compounds. The characterization of these clusters has represented a great source of genes for the generation of novel "unnatural natural" compounds by using combinatorial biosynthesis. The development of more effective methods for DNA sequencing, the improvement of targeted inactivation and heterologous host expression systems has strengthened the effectiveness of combinatorial biosynthesis. For these reasons combinatorial DNA technology has become during the last decade one of the most important approaches for generating chemical structural diversity and for increasing the number of potential useful compounds.

Further reading: Streptomyces: Molecular Biology and Biotechnology]]> ConferencesMolecular Biology ConferenceMolecular Biology ConferencesWed, 08 Sep 2010 10:44:11 -0400http://www.caister.com/molecular-biology-blog/files/molecular-biology-angiogenesis.html#unique-entry-id-64http://www.caister.com/molecular-biology-blog/files/molecular-biology-angiogenesis.html#unique-entry-id-64Birmingham, UK Further information
Understanding angiogenesis (the growth of new blood vessels) is crucial to reduce morbidity and mortality from cardiovascular disease, alleviate developmental problems, and maximise reparative tissue remodelling. Of particular interest are those conditions where vascular growth is excessive (e.g. tumours) or insufficient (e.g. ischaemia). This meeting will emphasise an integrative approach to unravelling the molecular and cellular basis of angiogenesis. The likely focus will be on the control by vascular cell surface receptors, intracellular signalling pathways, cell proliferation, cell-cell interaction, and the functional consequences for health and disease. Recent progress in both technical and conceptual approaches are improving our understanding, and this meeting aims to facilitate cross-fertilisation of ideas among workers in related fields, but disciplines that rarely have the chance to interact.
Suggested reading: Molecular Biology Books]]> ConferencesThu, 19 Aug 2010 07:04:13 -0400http://www.caister.com/molecular-biology-blog/files/genomic-instability-conference.html#unique-entry-id-63http://www.caister.com/molecular-biology-blog/files/genomic-instability-conference.html#unique-entry-id-63Yerevan, Armenia Further information
FEBS Advanced Lecture Course

Suggested reading: Genetics Books]]> RegulationVirologyFri, 13 Aug 2010 09:08:40 -0400http://www.caister.com/molecular-biology-blog/files/viral-micrornas.html#unique-entry-id-62http://www.caister.com/molecular-biology-blog/files/viral-micrornas.html#unique-entry-id-62from Jennifer L. Umbach and Bryan R. Cullen writing in Alphaherpesviruses: Molecular Virology:

MicroRNAs (miRNAs) are an extensive class of approx 22 nucleotide long regulatory RNAs expressed by all mammalian cells and also by several DNA viruses, including many members of the herpesvirus family. Using deep sequencing technology, it has now been demonstrated that Herpes Simplex Virus 1 (HSV-1) encodes at least eight viral miRNAs, seven of which are expressed in latently infected human neurons. Similarly, HSV-2 has also been shown to encode at least six miRNAs, four of which are evolutionarily conserved between HSV-2 and HSV-1. Perhaps surprisingly, varicella zoster virus does not appear to express any viral miRNAs in latently infected cells. A recent review discusses the potential functions of the currently known HSV-1 and HSV-2 miRNAs, focusing on a possible role in stabilizing viral latency in infected neurons.

Further reading: Alphaherpesviruses: Molecular Virology]]> VirologyWed, 04 Aug 2010 03:11:32 -0400http://www.caister.com/molecular-biology-blog/files/hsv-nuclear-egress.html#unique-entry-id-61http://www.caister.com/molecular-biology-blog/files/hsv-nuclear-egress.html#unique-entry-id-61from Joel D. Baines writing in Alphaherpesviruses: Molecular Virology:

In a process unique in biology, all herpesviruses obtain their initial virion envelope by budding through the inner nuclear membrane. In the most prominent model of virion egress, the envelope of the perinuclear virion then fuses with the luminal surface of the outer nuclear membrane, releasing the de-enveloped capsid into the cytosol for subsequent budding events. The pUL31/pUL34 protein complex is a major player in the initial budding event, and mediates several relevant functions including disruption of the nuclear lamina, recruitment of other viral proteins to the inner nuclear membrane and perinuclear virion, and budding of the nucleocapsid through the inner nuclear membrane.

Further reading: Alphaherpesviruses: Molecular Virology]]> VirologyWed, 04 Aug 2010 03:02:15 -0400http://www.caister.com/molecular-biology-blog/files/apoptosis-modulation-herpes.html#unique-entry-id-60http://www.caister.com/molecular-biology-blog/files/apoptosis-modulation-herpes.html#unique-entry-id-60Apoptosis Modulation During Herpes Simplex Virus Replication
from Christopher R. Cotter and John A. Blaho writing in Alphaherpesviruses: Molecular Virology:

Consequences of human herpes simplex virus (HSV) infection include the induction of apoptosis and the concomitant synthesis of proteins which act to prevent this process from killing the infected cell. Recent data has clarified our current understanding of the mechanisms of induction and prevention of apoptosis by HSV; which ultimately establishes a delicate balance of pro- and antiapoptotic modulating factors in infected cells. These findings emphasize the fact that modulation of apoptosis by HSV during infection is a multicomponent phenomenon involving a combination of viral and cellular factors.

Further reading: Alphaherpesviruses: Molecular Virology]]> MedicineDrugsFri, 30 Jul 2010 04:04:41 -0400http://www.caister.com/molecular-biology-blog/files/personalized-medicine.html#unique-entry-id-59http://www.caister.com/molecular-biology-blog/files/personalized-medicine.html#unique-entry-id-59from Müller, 2011

The pathogenesis of common diseases, such as metabolic diseases, is caused by the complex and individual interplay of many susceptibility genes, which necessitates both personalized diagnosis and therapy. Small-molecule drugs which adequately address the multiple tissue-specific target proteins affected probably will not become available in near future. In contrast, therapeutic proteins, such as growth factors and antibodies, specifically replacing or inactivating the corresponding susceptibility gene products, are currently being identified with increasing efficacy. However, the failure to be administered by the oral route and to reach the cytoplasm of the diseased cells typically prevents their therapeutic use. Recent developments suggest that these limitations may be overcome by encapsulation of therapeutic proteins into nanoparticles or their covalent modification with glycolipid (glycosylphosphatidylinositol, GPI) structures. These act as membrane anchors for so-called GPI-anchored proteins and direct certain attached passenger proteins from lipid raft areas of the plasma membrane via cytoplasmic lipid droplets into small vesicles. These leave the donor cells and transfer the GPI-anchored proteins into the cytoplasm of acceptor cells. This pathway may enable the transport of therapeutic proteins across the intestinal barrier into the circulation and eventually across the plasma membrane of the diseased target cells. For therapy, a number of challenges remains to be tackled, in particular, control of release from the GPI anchor which determines the pharmacokinetic and pharmacodynamic profiles. Together these findings nourish the hope that oral path finding to drug targets by encapsulation and covalent modification of therapeutic proteins may enable personalized therapy of common diseases read more ...

Curr. Issues Mol. Biol. (2011) 13: 13-24]]> VirologyWed, 28 Jul 2010 12:29:42 -0400http://www.caister.com/molecular-biology-blog/files/oncolytic-vectors-cancer.html#unique-entry-id-58http://www.caister.com/molecular-biology-blog/files/oncolytic-vectors-cancer.html#unique-entry-id-58Oncolytic HSV (oHSV) virotherapy is a promising new strategy for cancer therapy, converting a human pathogen into a therapeutic agent. This takes advantage of the biology of HSV, by introducing genetic alterations that limit virus replication and cytotoxicity to transformed cancer cells while making the virus non-permissive in normal cells. HSV encodes a large number of genes that are non-essential for growth in tissue culture cells, but are nevertheless important for growth in post-mitotic cells and for interfering with intrinsic antiviral and innate immune responses. Many of the cellular pathways regulating growth and antiviral responses are disrupted in cancer cells, which means that viral gene products allowing replication in normal cells are not necessary in cancer cells. In considering the development of an infectious agent for human use, safety is a critical consideration. Therefore mutations targeting cancer cells must be combined with mutations in genes that play important roles in vivo; causing pathogenicity, spread through the nervous system and other organs, latency and reactivation, and adaptive immune responses. This review will focus more on the virological aspects of oHSV vectors and less on the cancer cell target, and describe the multiple strategies and genes involved in generating oHSV vectors. However, it is important to bear in mind that the effect of different HSV mutations will be highly dependent upon the physiology of the particular type of cancer cell and tumor, and that each oHSV vector will be more effective in some tumor types, so that it is unlikely that any one oHSV will be optimal for all types of cancer.

Further reading: Alphaherpesviruses: Molecular Virology]]> VirologyWed, 28 Jul 2010 12:28:01 -0400http://www.caister.com/molecular-biology-blog/files/autophagy-and-viruses.html#unique-entry-id-57http://www.caister.com/molecular-biology-blog/files/autophagy-and-viruses.html#unique-entry-id-57Autophagy is a rapidly growing area of biomedical research with broad relevance to fields including microbiology, cell biology, immunology, cancer biology, and neurodegeneration. In infection and immunity, it is emerging as a pivotal pathway mediating direct pathogen degradation as well as for the development of robust innate and adaptive immune responses. Successful pathogens have evolved to either evade or harness the autophagy pathway to further their replication and pathogenesis. In a recent review the basic aspects of autophagy will be described, along with its role in cellular homeostasis, and the development of immunity. The primary focus is a survey of past and recent research defining the interplay of autophagy and the herpesviruses, with particular reference to immune evasion and pathogenesis.

Further reading: Alphaherpesviruses: Molecular Virology]]> VirologyRNARegulationWed, 28 Jul 2010 12:25:34 -0400http://www.caister.com/molecular-biology-blog/files/hsv-microrna.html#unique-entry-id-56http://www.caister.com/molecular-biology-blog/files/hsv-microrna.html#unique-entry-id-56MicroRNAs (miRNAs) are an extensive class of approx 22 nucleotide long regulatory RNAs expressed by all mammalian cells and also by several DNA viruses, including many members of the herpesvirus family. Using deep sequencing technology, it has now been demonstrated that Herpes Simplex Virus 1 (HSV-1) encodes at least eight viral miRNAs, seven of which are expressed in latently infected human neurons. Similarly, HSV-2 has also been shown to encode at least six miRNAs, four of which are evolutionarily conserved between HSV-2 and HSV-1. Perhaps surprisingly, varicella zoster virus does not appear to express any viral miRNAs in latently infected cells. A recent review discusses the potential functions of the currently known HSV-1 and HSV-2 miRNAs, focusing on a possible role in stabilizing viral latency in infected neurons.

Further reading: Alphaherpesviruses: Molecular Virology]]> Book ReviewWed, 21 Jul 2010 10:51:43 -0400http://www.caister.com/molecular-biology-blog/files/microbial-biopolymer-book-review.html#unique-entry-id-55http://www.caister.com/molecular-biology-blog/files/microbial-biopolymer-book-review.html#unique-entry-id-55I am pleased to provide the following excerpt from a book review of Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives:

"The authors of this comprehensive review are internationally accepted specialists in the field of using microorganisms as a cell factory for biopolymers or special precursors of these polymers ... The editor and the authors have produced an excellent up-to date compendium which is extremely useful for all students of biotechnology, engineering and scientists in the biotechnological and microbiological branches and is recommended for all biotechnological and microbial laboratories and enterprises in this field. It should be available in libraries at universities, research institutes and biotechnological companies and is further strongly recommended to all those who are interested in life sciences." from Uta Breuer (Halle, Germany) writing in Clean (2009) 37(6): 414 read more ...

Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives Edited by: Bernd H. A. Rehm ISBN: 978-1-904455-36-3 Publisher: Caister Academic Press Publication Date: January 2009 Cover: hardback "an excellent up-to date compendium ... strongly recommended" (Clean)

]]> ConferencesGenomicsMolecular Biology ConferenceMolecular Biology ConferencesMon, 19 Jul 2010 11:49:10 -0400http://www.caister.com/molecular-biology-blog/files/regulatory-genomics.html#unique-entry-id-54http://www.caister.com/molecular-biology-blog/files/regulatory-genomics.html#unique-entry-id-54November 16 - 20, 2010 5th Annual DREAM Reverse Engineering Challenges
New York, USA Further information
Held jointly with the 6th Annual RECOMB Satellite on Systems Biology, and the 7th Annual RECOMB Satellite on Regulatory Genomics. The goal of the meeting is to bring together computational and experimental scientists in the area of regulatory genomics and systems biology, to discuss current research directions, latest findings, and establish new collaborations towards a systems-level understanding of gene regulation. The meeting consists of keynote presentations, oral presentations selected from submitted manuscripts and 1-page abstracts, and posters presentations also selected from submitted abstracts. More than 500 participants attended last year\'s meeting, the vast majority of whom attended all three meetings, and we expect a similar interest this year.
Suggested reading: Molecular Biology Books]]> ConferencesMolecular Biology ConferenceMolecular Biology ConferencesMon, 19 Jul 2010 11:47:35 -0400http://www.caister.com/molecular-biology-blog/files/protein-conference.html#unique-entry-id-53http://www.caister.com/molecular-biology-blog/files/protein-conference.html#unique-entry-id-53November 14 - 19, 2010 ESF-EMBO Symposium on Molecular Perspectives on Protein-Protein Interactions
Sant Feliu de Guixols, Spain Further information
The conference aims to gather scientists from molecular cell biology, biochemistry, structural biology, biophysics and bioinformatics with the common interest to explore the immensely important field of protein-protein interactions. The particular focus of the conference will be on molecular aspects of protein-protein interactions. Topics will include theory and computation, thermodynamics & kinetics, intrinsically unstructured protein complexes, PPI in disease and drug development, protein interaction networks, signaling complexes, membrane protein complexes, emerging and single molecule techniques, evolution and design as well as large multi-protein complexes. Fundamental and applied problems in these fields will be discussed from an interdisciplinary perspective.
Suggested reading: Molecular Biology Books]]> BooksFri, 16 Jul 2010 11:29:31 -0400http://www.caister.com/molecular-biology-blog/files/nanotechnology-water.html#unique-entry-id-52http://www.caister.com/molecular-biology-blog/files/nanotechnology-water.html#unique-entry-id-52The new book on Nanotechnology in Water Treatment Applications edited by T. Eugene Cloete, Michele de Kwaadsteniet, Marelize Botes and J. Manuel López-Romero has been published read more ...

Nanotechnology in Water Treatment Applications Edited by: T. Eugene Cloete, Michele de Kwaadsteniet, Marelize Botes and J. Manuel López-Romero ISBN: 978-1-904455-66-0 Publisher: Caister Academic Press Publication Date: June 2010 Cover: hardback

read more ...]]> Molecular Biology ConferencesMolecular Biology ConferenceConferencesThu, 01 Jul 2010 05:55:46 -0400http://www.caister.com/molecular-biology-blog/files/micro-rna-conference-2010.html#unique-entry-id-51http://www.caister.com/molecular-biology-blog/files/micro-rna-conference-2010.html#unique-entry-id-51
Oxford, UK Further information
Fifth International MicroRNAs Europe 2010‚ Meeting
Suggested reading: RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity
]]> ConferencesMolecular Biology ConferencesThu, 01 Jul 2010 05:53:14 -0400http://www.caister.com/molecular-biology-blog/files/european-genomics-2010-meeting.html#unique-entry-id-50http://www.caister.com/molecular-biology-blog/files/european-genomics-2010-meeting.html#unique-entry-id-50
Oxford, UK Further information
First International EGM: European Genomics-2010- Meeting. Topics include Genomic Studies in plants and animal model organisms, Genotyping with various Technological Platforms, Discovery of Single Nucleotide Polymorphisms and Human Trait Genes, SNP Hybrid Arrays and Mapping Studies, Copy Number and Structural Variations, Genome Variation and Allele-specific Gene Expression, Haplotype Mapping and Variome Projects, Comparative Genomic hybridization , Global Variation in the human Genome and Disease causing variants, SNP Genotyping and real-time PCR, Disease-associated SNPs , Disease Genetics and Diagnostics, Exponential Quantitative Trait Loss.
Suggested reading: Molecular Biology Books]]> RNARegulationGeneticsTue, 29 Jun 2010 03:31:19 -0400http://www.caister.com/molecular-biology-blog/files/rna-silencing-in-plants.html#unique-entry-id-49http://www.caister.com/molecular-biology-blog/files/rna-silencing-in-plants.html#unique-entry-id-49RNA Silencing in Plants and the Role of Viral Suppressors
from Ana Giner, Juan Jose Lopez-Moya and Lorant Lakatos writing in RNA Interference and Viruses
The term RNA silencing refers to several pathways present in eukaryotic organisms that lead to the sequence specific elimination or functional blocking of RNAs with homology to double stranded RNAs (dsRNAs) that have previously triggered the mechanism. Besides playing important roles in developmental control, RNA silencing forms part of the defence against viruses in plants, acting as a potent antiviral mechanism. To escape from the RNA silencing-based defence, most plant viruses make use of different strategies, the most common relying in the action of viral proteins with the capacity to suppress RNA silencing. The characterization of these viral suppressors is providing useful insights to understand how RNA silencing works, revealing components and steps in the silencing pathways.

Further reading: Recent Advances in Plant Virology | RNA Interference and Viruses | RNA and the Regulation of Gene Expression]]> VirologyRegulationExpressionTue, 29 Jun 2010 03:13:24 -0400http://www.caister.com/molecular-biology-blog/files/protein-expression.html#unique-entry-id-48http://www.caister.com/molecular-biology-blog/files/protein-expression.html#unique-entry-id-48Plant Viral Vectors for Protein Expression
from Yuri Y. Gleba and Anatoli Giritch writing in Recent Advances in Plant Virology

Plant-virus-driven transient expression of heterologous proteins is the basis of several mature manufacturing processes that are currently being used for the production of multiple proteins including vaccine antigens and antibodies. Viral vectors have also become useful tools for research. In recent years, advances have been made both in the development of first-generation vectors (those that employ the 'full virus' strategy) as well as second-generation vectors designed using the 'deconstructed virus' approach. This second strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons. Among the most often used viral backbones are those of Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus. Prototypes of industrial processes that provide for high-yield, rapid scale-up, and fast manufacturing have been recently developed using viral vectors, with several manufacturing facilities compliant with good manufacturing practices (GMP) in place, and a number of pharmaceutical proteins currently in pre-clinical and clinical trials.

Further reading: Recent Advances in Plant Virology | Virology Publications]]> RNARegulationTue, 29 Jun 2010 03:10:44 -0400http://www.caister.com/molecular-biology-blog/files/rna-silencing.html#unique-entry-id-47http://www.caister.com/molecular-biology-blog/files/rna-silencing.html#unique-entry-id-47RNA Silencing and the Interplay Between Plants and Viruses
from Lourdes Fernández-Calvino, Livia Donaire and César Llave writing in Recent Advances in Plant Virology

In eukaryotes, RNA silencing controls gene expression to regulate development, genome stability and stress-induced responses. In plants, this process is also recognized as a major immune system targeted against plant viruses. Plant viruses stimulate RNA silencing responses though formation of viral RNA with double-stranded features that are subsequently processed into functional small RNAs (sRNAs). Recent studies highlight the complexity of the viral sRNA populations and their potential to associate with multiple silencing effector complexes. This fact has profound implications in the cross-talk interactions between plants and viruses since both virus genomes and host genes are putative targets of viral sRNAs. The concept of RNA silencing is an elegant natural antiviral mechanism in plants. Viral sRNA-mediated regulation of gene expression is important in the frame of compatible interactions between plants and viruses.

Further reading: Recent Advances in Plant Virology | Virology Publications | RNA and the Regulation of Gene Expression]]> VaccinesGenomicsThu, 24 Jun 2010 06:15:23 -0400http://www.caister.com/molecular-biology-blog/files/glycoconjugate-vaccine.html#unique-entry-id-46http://www.caister.com/molecular-biology-blog/files/glycoconjugate-vaccine.html#unique-entry-id-46Glycoconjugate Vaccine
from David R. Bundle writing in Vaccine Design: Innovative Approaches and Novel Strategies

Methods for single point attachment of polysaccharides and oligosaccharides to protein carriers and T-cell peptides are important in vaccine design. Contemporary approaches involve synthetic oligosaccharides with linker or tether chemistry designed for compatibility with synthetic strategies. Current research involves the synthesis and evaluation of conjugate vaccines designed to combat infectious bacterial and fungal diseases, as well as the design and testing of therapeutic cancer vaccine. The prevailing dogma that protective B-cell epitopes should be comprised of 10-20 monosaccharides is confirmed for several experimental vaccines including those directed toward Shigell flexneri and Shigella dysenteriae. However, several small epitopes composed of 3-5 monosaccharide residues are sufficient to induce antibody against the whole organism and to confer protection.

Further reading: Vaccine Design: Innovative Approaches and Novel Strategies]]> VaccinesGenomicsThu, 24 Jun 2010 06:04:23 -0400http://www.caister.com/molecular-biology-blog/files/genome-vaccines.html#unique-entry-id-45http://www.caister.com/molecular-biology-blog/files/genome-vaccines.html#unique-entry-id-45The first genome-derived vaccine now in clinical trials
from Fabio Bagnoli, Nathalie Norais, Ilaria Ferlenghi, Maria Scarselli, Claudio Donati, Silvana Savino, Michèle A. Barocchi and Rino Rappuoli writing in Vaccine Design: Innovative Approaches and Novel Strategies

Genome sequencing has become routine, and modern vaccine design is taking advantage of the accumulating genomic information. Reverse vaccinology is built on genome-based antigen discovery and has largely replaced classical vaccinology methods based on growing and dissecting the microorganism. The main advantage of the approach is the fast prediction of vaccine candidates. Most of the antigens will be surface exposed proteins, since these antigens are most likely accessible to antibodies. This approach can be applied to non-cultivable microorganisms, something difficult or impossible to do with conventional approaches. When the first reverse vaccinology project was started, in the year 2000, antigen identification was mainly based on bioinformatic analysis of one genome. Since then, the technique has shown its full potential, with the first genome-derived vaccine now in clinical trials and several vaccines in preclinical studies. In the meantime the approach has been improved with the support of proteomics, functional genomics and comparative genomics. The complete process includes antigen prediction to high-throughput purification, screening and selection of the vaccine composition.

Further reading: Vaccine Design: Innovative Approaches and Novel Strategies]]> Molecular Biology ConferenceWed, 23 Jun 2010 09:14:07 -0400http://www.caister.com/molecular-biology-blog/files/applied-microbiology-conference.html#unique-entry-id-44http://www.caister.com/molecular-biology-blog/files/applied-microbiology-conference.html#unique-entry-id-44Karlsruhe, Germany Further information
Main topics: Cell Biology, Environmental Microbiology, Food Microbiology, Microbial Interactions, New Imaging and other innovative Techniques, Stress Responses, White Biotechnology

Suggested reading: Microbiology Books]]> Molecular Biology ConferenceWed, 23 Jun 2010 09:12:51 -0400http://www.caister.com/molecular-biology-blog/files/white-biotechnology.html#unique-entry-id-43http://www.caister.com/molecular-biology-blog/files/white-biotechnology.html#unique-entry-id-43Bielefeld, Germany Further information
Chair: Volker Wendisch, Bielefeld University, Institut fur Genomforschung und Systembiologie, DE, Oluf Kruse, Bielefeld University, Center for Biotechnology. Closing date for application is 10th of September, 2010.
Suggested reading: Microbiology Books]]> Wed, 23 Jun 2010 09:11:57 -0400http://www.caister.com/molecular-biology-blog/files/microscopy-course.html#unique-entry-id-42http://www.caister.com/molecular-biology-blog/files/microscopy-course.html#unique-entry-id-42Heidelberg, Germany Further information
EMBO Practical Course

Suggested reading: Molecular Biology Books
]]> Molecular Biology ConferenceWed, 23 Jun 2010 09:10:51 -0400http://www.caister.com/molecular-biology-blog/files/protein-protein-interactions.html#unique-entry-id-41http://www.caister.com/molecular-biology-blog/files/protein-protein-interactions.html#unique-entry-id-41Sant Feliu de Guixols, Spain Further information
ESF-EMBO Symposium. Chaired by: Dr. Jacob Piehler, University of Osnabruck, DE, Co-Chairs: Gideon Schreiber, Weizmann Institute of Science, IL, Colin Kleanthous, University of York, UK
Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceWed, 23 Jun 2010 09:09:44 -0400http://www.caister.com/molecular-biology-blog/files/cytoskeleton.html#unique-entry-id-40http://www.caister.com/molecular-biology-blog/files/cytoskeleton.html#unique-entry-id-40Sant Feliu de Guixols, Spain Further information
Chaired by: Michelle Peckham, University of Leeds, Institute of Molecular and Cellular Biology, Centre for Human Biology, UK, Claudia Veigel, National Institute of Medical Research, Physical Biochemistry Department, UK

Suggested reading: Molecular Biology Books]]> Molecular Biology ConferenceWed, 23 Jun 2010 09:07:06 -0400http://www.caister.com/molecular-biology-blog/files/transcription-chromatin-conference.html#unique-entry-id-39http://www.caister.com/molecular-biology-blog/files/transcription-chromatin-conference.html#unique-entry-id-39Heidelberg, Germany Further information
9th EMBL Conference. Transcription and Chromatin. EMBL Courses and Conferences. Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.

Suggested reading: Molecular Biology Books
]]> BooksBook ReviewTue, 22 Jun 2010 09:06:48 -0400http://www.caister.com/molecular-biology-blog/files/abc-transport.html#unique-entry-id-38http://www.caister.com/molecular-biology-blog/files/abc-transport.html#unique-entry-id-38ABC Transporters in Microorganisms:

"of practical use to any scientist working on active transport systems whether in bacteria, parasites, or human cells. It is written in a fashion that allows readers to focus on specific topics and shows comparisons between systems. All the authors are from different disciplines but have contributed their knowledge to a cohesive book ... The book contains some excellent figures of the folding patterns of the proteins and the dynamics of how they change to import or export specific substrates ... well-organized and well-written book ... should be considered an essential reference for laboratories working in this area." from Rebecca T. Horvat (University of Kansas Medical Center, USA) writing in Doodys read more ...

ABC Transporters in Microorganisms Edited by: Alicia Ponte-Sucre ISBN: 978-1-904455-49-3 Publisher: Caister Academic Press Publication Date: August 2009 Cover: hardback "well-organized and well-written ... an essential reference" (Doodys)

]]> TechnologyRNAWed, 16 Jun 2010 11:11:43 -0400http://www.caister.com/molecular-biology-blog/files/small-rnas-salmonella.html#unique-entry-id-37http://www.caister.com/molecular-biology-blog/files/small-rnas-salmonella.html#unique-entry-id-37The small RNAs of Salmonella
from Sridhar Javayel, Kai Papenfort and Jörg Vogel writing in Salmonella: From Genome to Function

To date, close to one hundred distinct small noncoding RNAs (sRNAs) have been identified in Salmonella by a variety of biocomputational or wet-lab approaches including RNA sequencing. The function of more than twenty of these sRNAs is known from studies in Salmonella itself or can be inferred from conserved homologs in E. coli Many of these sRNAs act in conjunction with the RNA-chaperone Hfq to post-transcriptionally repress or activate trans-encoded target genes, but cis-antisense RNAs and regulators of protein activity are also abundantly present. In addition to a large number of sRNAs conserved in other enteric bacteria, Salmonella also expresses a set of sRNAs specific to this genus. Interestingly, such regulators have been shown to control the expression of conserved genes encoded on the "core" Salmonella genome. Conversely, conserved sRNA can act as regulators of recently acquired Salmonella-specific genes, indicating significant cross-talk of conserved and horizontally acquired elements at the RNA level. A recent review covers strategies for the identification of sRNAs as well as their characterized functional roles in Salmonella.

Further reading: Salmonella: From Genome to Function | RNA and the Regulation of Gene Expression]]> TechnologyWed, 16 Jun 2010 11:08:42 -0400http://www.caister.com/molecular-biology-blog/files/high-throughput-screening.html#unique-entry-id-36http://www.caister.com/molecular-biology-blog/files/high-throughput-screening.html#unique-entry-id-36High-throughput screening to determine the genetic requirements for Salmonella survival under different growth conditions
from Mollie Megan Reynolds, Rocio Canals, Michael McClelland and Helene Andrews-Polymenis writing in Salmonella: From Genome to Function

Salmonella species are capable of survival in a wide range of niches, both in the environment and in an infected host. Genetic requirements for survival of Salmonella in different niches have traditionally been identified using gene expression and forward genetics. The availability of complete genome sequences, microarray technology, and cost-effective new sequencing capabilities enabled increasingly efficient high-throughput analyses of Salmonella genomes to identify elements that contribute to survival in these niches. A recent review describes many of the high-throughput tools that have been developed over the past two decades, and the genetic requirements for Salmonella survival that have been identified using these techniques.

Further reading: Salmonella: From Genome to Function]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:30:17 -0400http://www.caister.com/molecular-biology-blog/files/microfluidic-pcr.html#unique-entry-id-33http://www.caister.com/molecular-biology-blog/files/microfluidic-pcr.html#unique-entry-id-33Microfluidic Emulsion PCR
from N. Reginald Beer and John H. Leamon writing in PCR Troubleshooting and Optimization: The Essential Guide

PCR has traditionally been performed in microliter-scale reactions because larger scale volumes are prohibitively expensive and wasteful while the smaller scales (nanoliter and below) are impractical with available sample handling tools and detection systems. At the microliter scale, samples can contain mutually competitive and distinct targets, introducing amplification bias and competitive inhibition that degrade assay performance. Microfluidic Emulsion PCR has emerged as a technique to resolve these challenges by a combination of two enabling technologies. Emulsion PCR provides the advantages of fluid partitioning, namely elimination of sample bias and the ability to run millions of reactions in discrete volumes, while microfluidics simultaneously reduces the sample volume, introduces a level of control over emulsion parameters, and provides optical observability of the partitioned microreactors. Furthermore, since microfluidic emulsions can be made monodisperse in size, they allow the assumption of an average dilution per reactor to permit the exploitation of Poisson statistics for very accurate titer estimation. Microfluidic emulsions can also be employed to perform solid-phase amplification with bead-based assays, combining yet another useful technique with the sample partitioning benefits of droplets. We expect the advantages of both emulsion PCR and microfluidics will encourage new applications and the integration of these enabling technologies will improve PCR performance.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:30:12 -0400http://www.caister.com/molecular-biology-blog/files/pcr-hmra.html#unique-entry-id-32http://www.caister.com/molecular-biology-blog/files/pcr-hmra.html#unique-entry-id-32High Resolution Melting Analysis
from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide

Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA) is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide
]]> PCRReal-Time PCRqPCRRT-PCREpigeneticsTue, 15 Jun 2010 10:30:09 -0400http://www.caister.com/molecular-biology-blog/files/pcr-epigenetics.html#unique-entry-id-31http://www.caister.com/molecular-biology-blog/files/pcr-epigenetics.html#unique-entry-id-31PCR Applications for Epigenetics Research
from Gavin Meredith, Miro Dudas, Mark Landers, Vasiliki Anest, Jonathan Wang, Caifu Chen, Peter Jozsi and Christopher Adams writing in PCR Troubleshooting and Optimization: The Essential Guide

The field of epigenetics transcends traditional genetics, genomics, molecular biology, and is poised to revolutionize the field of medical research and healthcare. It is a diverse field that encompasses the study of nuclear components such as chromatin structure, including histone modifications, protein/DNA interactions, protein/RNA interactions, and how these factors influence gene function. It also includes the study of DNA methylation and the role that non-coding RNAs play in influencing DNA methylation patterns, chromatin structure and ultimately regulating gene expression. Just as the field of epigenetics is broad and complex, so is the molecular technology of polymerase chain reaction (PCR). For every question one would like to address in any of these areas of epigenetics, there is a PCR application and instrumentation suitable to address it. For example there are numerous PCR-based approaches to look at DNA methylation patterns, densities, and even the methylation status of individual cytosine residues by PCR. Additionally, there are PCR methods to survey ncRNA expression and identify regions of the genome where proteins and RNA interact or where certain functional histone marks are located.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:30:07 -0400http://www.caister.com/molecular-biology-blog/files/pcr-miqe.html#unique-entry-id-30http://www.caister.com/molecular-biology-blog/files/pcr-miqe.html#unique-entry-id-30The MIQE Guidelines Uncloaked
from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide

The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:30:04 -0400http://www.caister.com/molecular-biology-blog/files/cfb490a444fad0bb2f7d75a00fd8b045-29.html#unique-entry-id-29http://www.caister.com/molecular-biology-blog/files/cfb490a444fad0bb2f7d75a00fd8b045-29.html#unique-entry-id-29qPCR Data Analysis: Unlocking the Secret to Successful Results
from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide

Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. Data analysis aspects include the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:30:02 -0400http://www.caister.com/molecular-biology-blog/files/pcr-instrumentation.html#unique-entry-id-28http://www.caister.com/molecular-biology-blog/files/pcr-instrumentation.html#unique-entry-id-28Real-Time PCR Instrumentation: An Instrument Selection Guide
from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide

A paper from 2008 mentions that quantitative PCR is 25 years old but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems. Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instrument available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:29:59 -0400http://www.caister.com/molecular-biology-blog/files/pcr-optimization.html#unique-entry-id-27http://www.caister.com/molecular-biology-blog/files/pcr-optimization.html#unique-entry-id-27RT-PCR Optimization Strategies
from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide

PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:29:56 -0400http://www.caister.com/molecular-biology-blog/files/pcr-sensitivity.html#unique-entry-id-26http://www.caister.com/molecular-biology-blog/files/pcr-sensitivity.html#unique-entry-id-26Obtaining Maximum PCR Sensitivity and Specificity
from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. Addressing all of these factors will aid the investigator in designing high quality PCR assays.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:29:11 -0400http://www.caister.com/molecular-biology-blog/files/pcr-controls.html#unique-entry-id-25http://www.caister.com/molecular-biology-blog/files/pcr-controls.html#unique-entry-id-25Significance of Controls and Standard Curves in PCR
from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:

Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper Optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRReal-Time PCRqPCRRT-PCRTue, 15 Jun 2010 10:27:42 -0400http://www.caister.com/molecular-biology-blog/files/pcr-inhibitors.html#unique-entry-id-24http://www.caister.com/molecular-biology-blog/files/pcr-inhibitors.html#unique-entry-id-24Difficult Templates and Inhibitors of PCR
from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:

One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay.

To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]> PCRTue, 15 Jun 2010 10:26:39 -0400http://www.caister.com/molecular-biology-blog/files/pcr-history.html#unique-entry-id-23http://www.caister.com/molecular-biology-blog/files/pcr-history.html#unique-entry-id-23A Brief History of PCR
from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:

The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.

Read more: PCR Troubleshooting and Optimization: The Essential Guide]]> ConferencesRNATue, 08 Jun 2010 03:50:21 -0400http://www.caister.com/molecular-biology-blog/files/non-coding-genome.html#unique-entry-id-22http://www.caister.com/molecular-biology-blog/files/non-coding-genome.html#unique-entry-id-22Heidelberg, Germany Further information

This symposium will provide an interdisciplinary discussion of the roles of non-coding RNAs with the aim of enhancing our understanding of gene regulation and function. Topics will include recent discoveries in the fields of prokaryotic and eukaryotic long and short non-coding RNAs. The functional roles of non-coding RNAs in a wide variety of cell processes will be discussed.
Suggested reading: RNA Interference and Viruses: Current Innovations and Future Trends]]> ConferencesTue, 08 Jun 2010 03:48:21 -0400http://www.caister.com/molecular-biology-blog/files/neural-circuits.html#unique-entry-id-21http://www.caister.com/molecular-biology-blog/files/neural-circuits.html#unique-entry-id-21Heidelberg, Germany Further information

Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.
Suggested reading: Molecular Biology Books]]> ConferencesPCRTechnologyMon, 07 Jun 2010 12:26:52 -0400http://www.caister.com/molecular-biology-blog/files/pcr-seminar-miqe.html#unique-entry-id-20http://www.caister.com/molecular-biology-blog/files/pcr-seminar-miqe.html#unique-entry-id-20
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.

This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event..

You can also click here to take a look at our archive for this series, which now contains:

Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer

Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125

Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh]]> BooksBiotechnologyGenomicsMon, 07 Jun 2010 07:21:41 -0400http://www.caister.com/molecular-biology-blog/files/streptomyces-book.html#unique-entry-id-19http://www.caister.com/molecular-biology-blog/files/streptomyces-book.html#unique-entry-id-19Streptomyces: Molecular Biology and Biotechnology
Streptomycetes are Gram-positive, high GC-description, sporulating bacteria found predominantly in soil. Streptomycetes are characterised by a complex secondary metabolism producing antibiotic compounds and other metabolites with medicinal properties. In recent years genomic studies, genomic mining and biotechnological approaches have been employed in the search for new antibiotics and other drugs.
With contributions from some of the leading scientists in the field, this volume documents recent research and development in streptomycetes genomics, physiology and metabolism. With a focus on biotechnology and genomics, the book provides an excellent source of up-to-date information. Topics include: genome architecture, conjugative genetic elements, differentiation, protein secretion, central carbon metabolic pathways, regulation of nitrogen assimilation, phosphate control of metabolism, gamma-butyrolactones and their role in antibiotic regulation, clavulanic acid and clavams, genome-guided exploration, gene clusters for bioactive natural products, genomics of cytochromes p450.

Streptomyces: Molecular Biology and Biotechnology Edited by: Paul Dyson ISBN: 978-1-904455-77-6 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback

Essential reading for research scientists, biotechnologists, graduate students and other professionals involved in streptomycetes research, antibiotic and antimicrobial development, drug discovery, soil microbiology and related fields. A recommended text for all microbiology laboratories.

• Magic in Solution: An Introduction and Brief History of PCR
  Speaker: Carl Wittwer
  18 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• Obtaining Maximum PCR Sensitivity and Specificity
  Speaker: Cameron N. Gundry
  25 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• Significance of Controls and Standard Curves in PCR
  Speaker: Ian Kavanagh
  01 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• The MBD2-based Enrichment Approach for Analyzing DNA methylation
  Speaker: Chris Adams
  08 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• The MIQE Guidelines Uncloaked
  Speaker: Greg Shipley
  15 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• High Resolution Melting Analysis - Beyond the SNP
  Speaker: John Mackay
  22 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET

• Bifidobacteria: Genomics and Molecular Aspects
• Lactobacillus Molecular Biology