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This unique and practical resource provides the most complete and concise summary of underlying principles and approaches to studying nucleic acid structure, including discussion of x-ray crystallography, NMR, molecular modelling, and databases.

Species-specific oligonucleotide primers for detecting wood rot fungi, Gloeophyllum trabeum, Trametes versicolor, Coniophora puteana, and Serpula lacrymans, and the primer detecting a group of related fungi to G. sepiarium were developed. These primer sequences were picked up from the internal transcribed spacer region between small-subunit rDNA and large-subunit rDNA. The species selectivities of the developed primers were checked. Real-time polymerase chain reaction (PCR) was carried out using these highly specific primers to quantitatively detect at least of 0.01 ng genome DNA of the target species. This quantitative PCR was also used to differentiate the target species DNA from mixed species DNA. A PCR-based technique using the species-specific primers would be applicable to multiple-sample assay in diagnosis of wood decay and to investigation of environmental fungal populations.

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.

While structure-function relationships of proteins have been studied for a long time, structural studies of RNA face additional challenges. Nevertheless, with the continuous discovery of novel RNA molecules with key cellular functions and of novel pathways and interaction networks, the need for structural information of RNA is still increasing. This volume provides an introduction into techniques to assess structure and folding of RNA. Each chapter explains the theoretical background of one technique, and illustrates possibilities and limitations in selected application examples.

Oxidative stress is implicated in the pathogenesis of several viral infections, including hepatitis, influenza, and AIDS. Dietary oxidative stress due to either selenium or vitamin E deficiency increases cardiac damage in mice infected with a myocarditic strain of coxsackievirus B3. Such dietary oxidative stress also allows a normally benign (i.e. amyocarditic) coxsackievirus B3 to convert to virulence and cause heart damage. This conversion to virulence is due to a nucleotide sequence change in the genome of the benign virus, which then resembles more closely the nucleotide sequence of virulent strains. Although it has been known for many years that poor nutrition can affect host response to infection, this is the first report of host nutrition affecting the genetic sequence of a pathogen. Further research is needed to determine whether poor host nutrition plays any role in the emergence of new vital diseases via alterations in the genotype of an infectious agent.

We developed a detection method for “Candidatus Liberibacter asiaticus”, causal agent of citrus huanglongbing, using isothermal and chimeric primer-initiated amplification of nucleic acids combined with cycling probe technology (Cycleave ICAN). With Cycleave ICAN, the reaction was done in one tube in 1 h without the need for electrophoresis, and false positives were not generated. In addition, Cycleave ICAN method was more sensitive than the conventional PCR method. Cycleave ICAN helps shorten the time for the large-scale detection needed to manage huanglongbing.

The BioWatch program, funded and overseen by the Department of Homeland Security (DHS), has three main elements-sampling, analysis, and response-each coordinated by different agencies. The Environmental Protection Agency maintains the sampling component, the sensors that collect airborne particles. The Centers for Disease Control and Prevention coordinates analysis and laboratory testing of the samples, though testing is actually carried out in state and local public health laboratories. Local jurisdictions are responsible for the public health response to positive findings. The Federal Bureau of Investigation is designated as the lead agency for the law enforcement response if a bioterrorism event is detected. In 2003 DHS deployed the first generation of BioWatch air samplers. The current version of this technology, referred to as Generation 2.0, requires daily manual collection and testing of air filters from each monitor. DHS has also considered newer automated technologies (Generation 2.5 and Generation 3.0) which have the potential to produce results more quickly, at a lower cost, and for a greater number of threat agents. Technologies to Enable Autonomous Detection for BioWatch is the summary of a workshop hosted jointly by the Institute of Medicine and the National Research Council in June 2013 to explore alternative cost-effective systems that would meet the requirements for a BioWatch Generation 3.0 autonomous detection system, or autonomous detector, for aerosolized agents . The workshop discussions and presentations focused on examination of the use of four classes of technologies-nucleic acid signatures, protein signatures, genomic sequencing, and mass spectrometry-that could reach Technology Readiness Level (TRL) 6-plus in which the technology has been validated and is ready to be tested in a relevant environment over three different tiers of temporal timeframes: those technologies that could be TRL 6-plus ready as part of an integrated system by 2016, those that are likely to be ready in the period 2016 to 2020, and those are not likely to be ready until after 2020. Technologies to Enable Autonomous Detection for BioWatch discusses the history of the BioWatch program, the role of public health officials and laboratorians in the interpretation of BioWatch data and the information that is needed from a system for effective decision making, and the current state of the art of four families of technology for the BioWatch program. This report explores how the technologies discussed might be strategically combined or deployed to optimize their contributions to an effective environmental detection capability.

Apple dimple fruit viroid (ADFVd) causes a severe fruit disorder in several apple cultivars. The viroid was discovered a few years ago and only a limited number of sequence variants has been reported so far. In this work, the sequence variability of two ADFVd field isolates from two commercial apple cultivars was studied. Sequencing of 18 full-length cDNA clones revealed five new sequence variants, all adopting a similar quasi-rod-like secondary structure of lowest free energy. Sequence comparison showed nine polymorphic positions distributed in different regions of the ADFVd molecule. Since symptoms of apple dimple fruit disease resemble those of dapple apple induced by Apple scar skin viroid (ASSVd) and it has been experimentally demonstrated that these two viroids may co-exist in the same plant, a fast and sensitive method for their rapid detection and discrimination is needed. We report here a method that meets such needs. Total nucleic acid minipreparations from symptomatic fruits, obtained by extraction with buffer-saturated phenol and further treatment with a silica-gel capture system, were used as templates for the simultaneous detection of ADFVd and ASSVd by multiplex fluorescent RT-PCR amplification. The incorporation in the PCR reaction of two viroid-specific primers, each labelled with a different fluorescent dye, simplifies and facilitates the distinction of the amplified products and avoids the use of the mutagenic and cancer inducing agent ethidium bromide for gel staining [Il Viroide dell'infossatura crateriforme della mela (ADFVd) causa gravi danni a molte cultivar di melo. Il viroide e' stato scoperto pochi anni fa ed e' stato riportato solo un limitato numero di sequenze. In questo lavoro e' stata studiata la variabilita' delle sequenze in due ceppi isolati da due cultivar commerciali. Siccome i sintomi di ADFVd assomigliano a quelli indotti dal Viroide dell'ulcerazione dell'epidermide delle mele (ASSVd) ed e' stato dimostrato che questi due viroidi possono coesistere nella stessa pianta, e' necessario un metodo sensibile per la rapida individuazione e discriminazione. Riportiamo qui il metodo che risponde a questa esigenza. Minipreparazioni di acido nucleico totale da frutti con sintomi, ottenute tramite un'estrazione con soluzione tampone di fenolo saturato e successivi trattamenti con un sistema di cattura al gel di silice, sono stati usati per una simultanea distinzione di ADFVd e di ASSVd, con una amplificazione RT-PCR multiplex in fluorescenza. Questo metodo semplifica e facilita la distinzione ed evita l'uso di agenti mutageni e cancerogeni come l'etidio bromuro]