Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat.

Expressed sequence tags (ESTs) are a valuable source of molecular markers. To enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in hexaploid wheat (Triticum aestivum L.), over 260,000 ESTs from 5 different grass species were analyzed and 5418 SSR-containing sequences were identified. Using sequence similarity analysis, 156 cross-species superclusters and 138 singletons were used to develop primer pairs, which were then tested on the genomic DNA of barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and wheat. Three-hundred sixty-eight primer pairs produced PCR amplicons from at least one species and 227 primer pairs amplified DNA from two or more species. EST-SSR sequences containing dinucleotide motifs were significantly more polymorphic (74%) than those containing trinucleotides (56%), and polymorphism was similar for markers in both coding and 5' untranslated (UTR) regions. Out of 112 EST-SSR markers, 90 identified 149 loci that were integrated into a reference wheat genetic map. These loci were distributed on 19 of the 21 wheat chromosomes and were clustered in the distal chromosomal regions. Multiple-loci were detected by 39% of the primer pairs. Of the 90 mapped ESTs, putative functions for 22 were identified using BLASTX queries. In addition, 80 EST-SSR markers (104 loci) were located to chromosomes using nullisomic-tetrasomic lines. The enhanced map from this study provides a basis for comparative mapping using orthologous and PCR-based markers and for identification of expressed genes possibly affecting important traits in wheat.     

Nanodiagnostic Method for Colorimetric Detection of Mycobacterium tuberculosis 16S rRNA

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA–DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml^?1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.     

Metalloproteinases and their inhibitors: regulators of wound healing

Wound healing is a dynamic process that involves a coordinated response of many cell types representing distinct tissue compartments and is fundamentally similar among tissue types. Among the many gene products that are essential for restoration of normal tissue architecture, several members of the matrix metalloproteinase (MMP) family function as positive and, at times, negative regulators of repair processes. MMPs were initially thought to only function in the resolution phase of wound healing, particularly during scar resorption; however, recent evidence suggests that they also influence other wound-healing responses, such as inflammation and re-epithelialization. In this review, we discuss what is currently known about the function of MMPs in wound healing and will provide suggestions for future research directions.     

The reliability and validity of fatigue measures during multiple-sprint work: an issue revisited

The ability to repeatedly produce a high-power output or sprint speed is a key fitness component of most field and court sports. The aim of this study was to evaluate the validity and reliability of eight different approaches to quantify this parameter in tests of multiple-sprint performance. Ten physically active men completed two trials of each of two multiple-sprint running protocols with contrasting recovery periods. Protocol 1 consisted of 12 x 30-m sprints repeated every 35 seconds; protocol 2 consisted of 12 x 30-m sprints repeated every 65 seconds. All testing was performed in an indoor sports facility, and sprint times were recorded using twin-beam photocells. All but one of the formulae showed good construct validity, as evidenced by similar within-protocol fatigue scores. However, the assumptions on which many of the formulae were based, combined with poor or inconsistent test-retest reliability (coefficient of variation range: 0.8-145.7%; intraclass correlation coefficient range: 0.09-0.75), suggested many problems regarding logical validity. In line with previous research, the results support the percentage decrement calculation as the most valid and reliable method of quantifying fatigue in tests of multiple-sprint performance.     

Genome-scale analysis of anti-metabolite directed strain engineering

Classic strain engineering methods have previously been limited by the low-throughput of conventional sequencing technology. Here, we applied a new genomics technology, scalar analysis of library enrichments (SCALEs), to measure >3 million Escherichia coli genomic library clone enrichment patterns resulting from growth selections employing three aspartic-acid anti-metabolites. Our objective was to assess the extent to which access to genome-scale enrichment patterns would provide strain-engineering insights not reasonably accessible through the use of conventional sequencing. We determined that the SCALEs method identified a surprisingly large range of anti-metabolite tolerance regions (423, 865, or 909 regions for each of the three anti-metabolites) when compared to the number of regions (1-3 regions) indicated by conventional sequencing. Genome-scale methods uniquely enable the calculation of clone fitness values by providing concentration data for all clones within a genomic library before and after a period of selection. We observed that clone fitness values differ substantially from clone concentration values and that this is due to differences in overall clone fitness distributions for each selection. Finally, we show that many of the clones of highest fitness overlapped across all selections, suggesting that inhibition of aspartate metabolism, as opposed to specific inhibited enzymes, dominated each selection. Our follow up studies confirmed our observed growth phenotypes and showed that intracellular amino-acid levels were also altered in several of the identified clones. These results demonstrate that genome-scale methods, such as SCALEs, can be used to dramatically improve understanding of classic strain engineering approaches.     

Genomic targeting and mapping of tiller inhibition gene (<Emphasis Type="Italic">tin3</Emphasis>) of wheat using ESTs and synteny with rice

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3A^mL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat–rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micro-colinearity between rice, <Emphasis Type="Italic">Brachypodium</Emphasis>, and <Emphasis Type="Italic">Triticum monococcum</Emphasis> at the wheat domestication locus <Emphasis Type="Italic">Q</Emphasis>

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.     

A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread

Viruses lack self-propulsion. To move in multi-cellular hosts they must therefore manipulate infected cells. Herpesviruses provide an archetype for many aspects of host manipulation, but only for alpha-herpesviruses in is there much information about they move. Other herpesviruses are not necessarily the same. Here we show that Murine gamma-herpesvirus-68 (MHV-68) induces the outgrowth of long, branched plasma membrane fronds to create an intercellular network for virion traffic. The fronds were actin-based and RhoA-dependent. Time-lapse imaging showed that the infected cell surface became highly motile and that virions moved on the fronds. This plasma membrane remodelling was driven by the cytoplasmic tail of gp48, a MHV-68 glycoprotein previously implicated in intercellular viral spread. The MHV-68 ORF58 was also required, but its role was simply transporting gp48 to the plasma membrane, since a gp48 mutant exported without ORF58 did not require ORF58 to form membrane fronds either. Together, gp48/ORF58 were sufficient to induce fronds in transfected cells, as were the homologous BDLF2/BMRF2 of Epstein-Barr virus. Gp48/ORF58 therefore represents a conserved module by which gamma-herpesviruses rearrange cellular actin to increase intercellular contacts and thereby promote their spread.     

Effects of spring temperature and volcanic eruptions on wader productivity

Key demographic parameters often show substantial annual variation that can have important consequences for rates of population growth. Since 2011 we have conducted annual estimates of the productivity of Icelandic Black‐tailed Godwits Limosa limosa islandica over a large part of their breeding range. During this period, a volcanic eruption resulted in extensive dust deposition across the region. We show that Godwit productivity varies with spring temperatures but in the year of the volcanic eruption, productivity was reduced to almost zero. This rare but extreme event is likely to have had only a short‐term influence, whereas ongoing warming of sub‐Arctic regions is potentially a more substantial driver of the continued growth of this population.