Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Introduction: Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain. However, limited natural variation and the proximity of these genes complicates the selection of extremely low-PPO wheat varieties by recombination. The goal of the current study was to edit all copies of PPO1 and PPO2 to drive extreme reductions in PPO grain activity in elite wheat varieties. Results: A CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar ‘Fielder’. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type. The same construct was transformed into the elite winter wheat cultivars ‘Guardian’ and ‘Steamboat’, which have five PPO1 and PPO2 genes. In these varieties PPO activity was reduced by >90% in both T1 and T2 lines. In all three varieties, dough samples from edited lines exhibited reduced browning. Discussion: This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in cropbreeding programs by inducing novel variation in loci inaccessible to recombinatio

Suppression of Phase Separation in LiFePO4 Nanoparticles During Battery Discharge

Using a novel electrochemical phase-field model, we question the common belief that LixFePO4 nanoparticles separate into Li-rich and Li-poor phases during battery discharge. For small currents, spinodal decomposition or nucleation leads to moving phase boundaries. Above a critical current density (in the Tafel regime), the spinodal disappears, and particles fill homogeneously, which may explain the superior rate capability and long cycle life of nano-LiFePO4 cathodes.Comment: 27 pages, 8 figure

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASNB2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality

Background: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine levels, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. Results: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine levels in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high levels as a result of sulphur deficiency. Conclusions: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine levels in commercial wheat grain

Review: optical fiber sensors for civil engineering applications

Optical fiber sensor (OFS) technologies have developed rapidly over the last few decades, and various types of OFS have found practical applications in the field of civil engineering. In this paper, which is resulting from the work of the RILEM technical committee “Optical fiber sensors for civil engineering applications”, different kinds of sensing techniques, including change of light intensity, interferometry, fiber Bragg grating, adsorption measurement and distributed sensing, are briefly reviewed to introduce the basic sensing principles. Then, the applications of OFS in highway structures, building structures, geotechnical structures, pipelines as well as cables monitoring are described, with focus on sensor design, installation technique and sensor performance. It is believed that the State-of-the-Art review is helpful to engineers considering the use of OFS in their projects, and can facilitate the wider application of OFS technologies in construction industry

Genomic-based epidemiology reveals independent origins and gene flow of glyphosate resistance in Bassia scoparia populations across North America

Genomic-based epidemiology can provide insight into the origins and spread of herbicide resistance mechanisms in weeds. We used kochia (Bassia scoparia) populations resistant to the herbicide glyphosate from across western North America to test the alternative hypotheses that (i) a single EPSPS gene duplication event occurred initially in the Central Great Plains and then subsequently spread to all other geographical areas now exhibiting glyphosate-resistant kochia populations or that (ii) gene duplication occurred multiple times in independent events in a case of parallel evolution. We used qPCR markers previously developed for measuring the structure of the EPSPS tandem duplication to investigate whether all glyphosate-resistant individuals had the same EPSPS repeat structure. We also investigated population structure using simple sequence repeat markers to determine the relatedness of kochia populations from across the Central Great Plains, Northern Plains and the Pacific Northwest. We found that the original EPSPS duplication genotype was predominant in the Central Great Plains where glyphosate resistance was first reported. We identified two additional EPSPS duplication genotypes, one having geographical associations with the Northern Plains and the other with the Pacific Northwest. The EPSPS duplication genotype from the Pacific Northwest seems likely to represent a second, independent evolutionary origin of a resistance allele. We found evidence of gene flow across populations and a general lack of population structure. The results support at least two independent evolutionary origins of glyphosate resistance in kochia, followed by substantial and mostly geographically localized gene flow to spread the resistance alleles into diverse genetic backgrounds

Daphniaperformance on diets containing different combinations of high-quality algae, heterotrophic bacteria, and allochthonous particulate organic matter

Filter-feeding zooplankton in lakes feed on a mixture of phytoplankton, bacteria, and terrestrial particles and the proportions and nutritional value of these components can be highly variable. However, the extent to which food quality interacts with food quantity in affecting overall zooplankton performance is not yet fully resolved. Here we performed laboratory feeding experiments to test how the performance of the unselective filter feederDaphnia galeatawas affected if various quantities of high-quality food (the phytoplanktonRhodomonas) were diluted with low-quality food such as heterotrophic bacteria (Pseudomonas) or terrestrial detritus particles (t-POM) from the riparian zone of a boreal forest stream. We hypothesised: that increased proportions of bacteria and t-POM in the diet will lead to decreased survival, somatic growth; and reproduction ofDaphniadespite the presence of phytoplankton; that these effects are more pronounced for t-POM than for heterotrophic bacteria; and that this response is stronger when phytoplankton availability is low. Increasing the concentrations ofPseudomonasaffectedDaphniasurvival, growth, and reproduction negatively whenRhodomonaswas available at intermediate (0.37 mgC/L) and high (0.55 mgC/L) quantities. WhenRhodomonasquantity was low (0.22 mgC/L), the addition ofPseudomonasgenerally resulted in betterDaphniaperformance except at very high concentrations of the bacterium relative toRhodomonas. In contrast, the addition of t-POM was detrimental for overallDaphniaperformance at allRhodomonasconcentrations. Daphniaperformance was best described by a model including the interaction between food quality and quantity, with stronger negative effects onDaphniawhen high-quality food was supplemented with t-POM than withPseudomonas. The results indicate that the ability of zooplankton to use low-quality food is affected by the concurrent availability of high-quality food. Furthermore, food sources that can be used but do not fulfil dietary requirements of grazers (e.g. bacteria), may still provide nutritional benefits as long as other complementary food components are available in sufficient quantities to compensate for biochemical deficiencies. Therefore, we conclude that heterotrophic bacteria, but not peat layer t-POM, can be an important component of zooplankton diets in boreal lakes, especially if the concentration of phytoplankton is low

Possible roles of Epstein-Barr virus in Castleman disease

<p>Abstract</p> <p>Background</p> <p>Complete resection seemed to be curative in patients with Castleman disease of any location but the disease is likely to be reactive in its pathogenesis. The relation between Epstein-Barr virus and Castleman disease has not been elucidated. We tried to define the role of Epstein-Barr virus in the pathogenesis of Castleman disease.</p> <p>Methods</p> <p>20 cases of Castleman disease were retrospectively reviewed from 1993 to 2006. At least 2 to 4 representative sections of formalin-fixed, paraffin-embedded specimens from each patient were obtained to examine the presence of EBV and its localization by hematoxylin-eosin stain, immunohistochemistry, polymerase chain reaction and In-situ hybridization</p> <p>Results</p> <p>Hyaline-vascular type was diagnosed in 18 cases, plasma cell type in 1 and mixed type in 1 case. All of them were positive for Epstein-Barr virus confirmed by PCR. For tumors that EBER(Epstein-Barr early region) signals mainly localized in the germinal centers have increased vascularity than cases with EBER detected in inter-follicular areas.</p> <p>Conclusion</p> <p>There is a strong association between Castleman disease and Epstein-Barr virus. EBV may have a potential role in angiogenesis of Castleman disease. For smaller lesion with high activity of angiogenesis but not amenable for curative resection, anti-angiogenesis medications may have a potential role to control the disease.</p

Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID

Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale