scholarly journals Functional dissection of HGGT and HPT in barley vitamin E biosynthesis via CRISPR/Cas9-enabled genome editing

2020 ◽  
Vol 126 (5) ◽  
pp. 929-942 ◽  
Author(s):  
Zhanghui Zeng ◽  
Ning Han ◽  
Cuicui Liu ◽  
B Buerte ◽  
Chenlu Zhou ◽  
...  

Abstract Background and Aims Vitamin E (tocochromanol) is a lipid-soluble antioxidant and an essential nutrient for human health. Among cereal crops, barley (Hordeum vulgare) contains a high level of vitamin E, which includes both tocopherols and tocotrienols. Although the vitamin E biosynthetic pathway has been characterized in dicots, such as Arabidopsis, which only accumulate tocopherols, knowledge regarding vitamin E biosynthesis in monocots is limited because of the lack of functional mutants. This study aimed to obtain gene knockout mutants to elucidate the genetic control of vitamin E composition in barley. Methods Targeted knockout mutations of HvHPT and HvHGGT in barley were created with CRISPR/Cas9-enabled genome editing. High-performance liquid chromatography (HPLC) was performed to analyse the content of tocochromanol isomers in transgene-free homozygous Hvhpt and Hvhggt mutants. Key Results Mutagenesis efficiency among T0 regenerated plantlets was 50–65 % as a result of two simultaneously expressed guide RNAs targeting each gene; most of the mutations were stably inherited by the next generation. The transgene-free homozygous mutants of Hvhpt and Hvhggt exhibited decreased grain size and weight, and the HvHGGT mutation led to a shrunken phenotype and significantly lower total starch content in grains. HPLC analysis revealed that targeted mutation of HvHPT significantly reduced the content of both tocopherols and tocotrienols, whereas mutations in HvHGGT completely blocked tocotrienol biosynthesis in barley grains. Transient overexpression of an HvHPT homologue in tobacco leaves significantly increased the production of γ- and δ-tocopherols, which may partly explain why targeted mutation of HvHPT in barley grains did not eliminate tocopherol production. Conclusions Our results functionally validated that HvHGGT is the only committed gene for the production of tocotrienols, whereas HvHPT is partly responsible for tocopherol biosynthesis in barley.

Author(s):  
Darya Meshalkina ◽  
Aleksei Glushchenko ◽  
Elana Kysil ◽  
Igor Mizgirev ◽  
Andrej Frolov

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.


2020 ◽  
Author(s):  
James McDonagh ◽  
William Swope ◽  
Richard L. Anderson ◽  
Michael Johnston ◽  
David J. Bray

Digitization offers significant opportunities for the formulated product industry to transform the way it works and develop new methods of business. R&D is one area of operation that is challenging to take advantage of these technologies due to its high level of domain specialisation and creativity but the benefits could be significant. Recent developments of base level technologies such as artificial intelligence (AI)/machine learning (ML), robotics and high performance computing (HPC), to name a few, present disruptive and transformative technologies which could offer new insights, discovery methods and enhanced chemical control when combined in a digital ecosystem of connectivity, distributive services and decentralisation. At the fundamental level, research in these technologies has shown that new physical and chemical insights can be gained, which in turn can augment experimental R&D approaches through physics-based chemical simulation, data driven models and hybrid approaches. In all of these cases, high quality data is required to build and validate models in addition to the skills and expertise to exploit such methods. In this article we give an overview of some of the digital technology demonstrators we have developed for formulated product R&D. We discuss the challenges in building and deploying these demonstrators.<br>


2020 ◽  
Vol 10 (3) ◽  
pp. 228-236 ◽  
Author(s):  
Lamia Taouzinet ◽  
Sofiane Fatmi ◽  
Allaeddine Khellouf ◽  
Mohamed Skiba ◽  
Mokrane Iguer-ouada

Background: Alpha-tocopherol is a potent antioxidant involved in sperm protection particularly during cryopreservation. However, its poor solubility limits the optimal protection in aqueous solutions. Objective: The aim of this study was to enhance the solubility of α-tocopherol by the use of liposomes. Methods: The experimental approach consisted to load vitamin E in liposomes prepared by ethanol injection method and the optimization carried out by an experimental design. The optimum solution was characterized by high performance liquid chromatography and scanning electron microscope. Finely, the impact on sperm motility protection was studied by the freezing technic of bovine sperm. Results: The optimum solution was obtained when using 10.9 mg/ml of phospholipids, 1.7 mg/ml of cholesterol and 2 mg/ml of vitamin E. The liposome size was 99.86 nm, providing 78.47% of loaded efficiency. The results showed also a significant positive impact on sperm motility after hours of preservation. Conclusion: In conclusion, the current results showed the interest of liposome preparation as an alternative to enhance vitamin E solubility and to protect spermatozoa during cryopreservation.


2020 ◽  
Vol 12 (2) ◽  
pp. 19-50 ◽  
Author(s):  
Muhammad Siddique ◽  
Shandana Shoaib ◽  
Zahoor Jan

A key aspect of work processes in service sector firms is the interconnection between tasks and performance. Relational coordination can play an important role in addressing the issues of coordinating organizational activities due to high level of interdependence complexity in service sector firms. Research has primarily supported the aspect that well devised high performance work systems (HPWS) can intensify organizational performance. There is a growing debate, however, with regard to understanding the “mechanism” linking HPWS and performance outcomes. Using relational coordination theory, this study examines a model that examine the effects of subsets of HPWS, such as motivation, skills and opportunity enhancing HR practices on relational coordination among employees working in reciprocal interdependent job settings. Data were gathered from multiple sources including managers and employees at individual, functional and unit levels to know their understanding in relation to HPWS and relational coordination (RC) in 218 bank branches in Pakistan. Data analysis via structural equation modelling, results suggest that HPWS predicted RC among officers at the unit level. The findings of the study have contributions to both, theory and practice.


2021 ◽  
pp. 1-7
Author(s):  
Haniel Fernandes

<b><i>Background:</i></b> Soccer is an extremely competitive sport, where the most match important moments can be defined in detail. Use of ergogenic supplements can be crucial to improve the performance of a high-performance athlete. Therefore, knowing which ergogenic supplements are important for soccer players can be an interesting strategy to maintain high level in this sport until final and decisive moments of the match. In addition, other supplements, such as dietary supplements, have been studied and increasingly referenced in the scientific literature. But, what if ergogenic supplements were combined with dietary supplements? This review brings some recommendations to improve performance of soccer athletes on the field through dietary and/or ergogenic supplements that can be used simultaneously. <b><i>Summary:</i></b> Soccer is a competitive sport, where the match important moments can be defined in detail. Thus, use of ergogenic supplements covered in this review can improve performance of elite soccer players maintaining high level in the match until final moments, such as creatine 3–5 g day<sup>−1</sup>, caffeine 3–6 mg kg<sup>−1</sup> BW around 60 min before the match, sodium bicarbonate 0.1–0.4 g kg<sup>−1</sup> BW starting from 30 to 180 min before the match, β-alanine 3.2 and 6.4 g day<sup>−1</sup> provided in the sustained-release tablets divided into 4 times a day, and nitrate-rich beetroot juice 60 g in 200 mL of water (6 mmol of NO3<sup>−</sup> L) around 120 min before match or training, including a combination possible with taurine 50 mg kg<sup>−1</sup> BW day<sup>−1</sup>, citrulline 1.2–3.4 g day<sup>−1</sup>, and arginine 1.2–6 g day<sup>−1</sup>. <b><i>Key Messages:</i></b> Soccer athletes can combine ergogenic and dietary supplements to improve their performance on the field. The ergogenic and dietary supplements used in a scientifically recommended dose did not demonstrate relevant side effects. The use of various evidence-based supplements can add up to further improvement in the performance of the elite soccer players.


Diversity ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 71
Author(s):  
Charalampos Dimitriadis ◽  
Ivoni Fournari-Konstantinidou ◽  
Laurent Sourbès ◽  
Drosos Koutsoubas ◽  
Stelios Katsanevakis

Understanding the interactions among invasive species, native species and marine protected areas (MPAs), and the long-term regime shifts in MPAs is receiving increased attention, since biological invasions can alter the structure and functioning of the protected ecosystems and challenge conservation efforts. Here we found evidence of marked modifications in the rocky reef associated biota in a Mediterranean MPA from 2009 to 2019 through visual census surveys, due to the presence of invasive species altering the structure of the ecosystem and triggering complex cascading effects on the long term. Low levels of the populations of native high-level predators were accompanied by the population increase and high performance of both native and invasive fish herbivores. Subsequently the overgrazing and habitat degradation resulted in cascading effects towards the diminishing of the native and invasive invertebrate grazers and omnivorous benthic species. Our study represents a good showcase of how invasive species can coexist or exclude native biota and at the same time regulate or out-compete other established invaders and native species.


Author(s):  
Umar Ibrahim Minhas ◽  
Roger Woods ◽  
Georgios Karakonstantis

AbstractWhilst FPGAs have been used in cloud ecosystems, it is still extremely challenging to achieve high compute density when mapping heterogeneous multi-tasks on shared resources at runtime. This work addresses this by treating the FPGA resource as a service and employing multi-task processing at the high level, design space exploration and static off-line partitioning in order to allow more efficient mapping of heterogeneous tasks onto the FPGA. In addition, a new, comprehensive runtime functional simulator is used to evaluate the effect of various spatial and temporal constraints on both the existing and new approaches when varying system design parameters. A comprehensive suite of real high performance computing tasks was implemented on a Nallatech 385 FPGA card and show that our approach can provide on average 2.9 × and 2.3 × higher system throughput for compute and mixed intensity tasks, while 0.2 × lower for memory intensive tasks due to external memory access latency and bandwidth limitations. The work has been extended by introducing a novel scheduling scheme to enhance temporal utilization of resources when using the proposed approach. Additional results for large queues of mixed intensity tasks (compute and memory) show that the proposed partitioning and scheduling approach can provide higher than 3 × system speedup over previous schemes.


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