CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry

Background: Gene editing using CRISPR/Cas9 is a simple and powerful tool for elucidating genetic controls and for crop improvement and its use has been reported in a growing number of important food crops, including recently Fragaria. In order to inform application of the technology in Fragaria, we targeted the visible endogenous marker gene PDS (phytoene desaturase) in diploid Fragaria vesca ssp. vesca ‘Hawaii 4’ and octoploid F. × ananassa ‘Calypso’. Results: Agrobacterium-mediated transformation of leaf and petiole explants was used for efficient stable integration of constructs expressing plant codon-optimised Cas9 and single guide sequences under control of the Arabidopsis U6-26 consensus promoter and terminator or Fragaria vesca U6III regulatory sequences. More than 80% (‘Hawaii 4’) and 50% (‘Calypso’) putative transgenic shoot lines (multiple shoots derived from a single callus) exhibited mutant phenotypes. Of mutant shoot lines selected for molecular analysis, approximately 75% (‘Hawaii 4’) and 55% (‘Calypso’) included albino regenerants with bi-allelic target sequence variants. Our results indicate the PDS gene is functionally diploid in ‘Calypso’. Conclusion: We demonstrate that CRISPR/Cas9 may be used to generate biallelic mutants at high frequency within the genomes of diploid and octoploid strawberry. The methodology, observations and comprehensive data set pre- sented will facilitate routine application of this technology in Fragaria to single and multiple gene copy targets where mutant phenotypes cannot be identified visually

ASSESSMENT OF LOWER LIMB ASYMMETRY: DIFFERENCES DURING ISOMETRIC AND STRETCH-SHORTENING CYCLE TASKS

Assessment of limb function asymmetry is of interest to practitioners due to its role in return to play guidelines and reported links to injury. Dynamometry is one of the most commonly used methods of assessing muscle function, but the mode of contraction used is different to the stretch-shortening cycles experienced during real-life activities. The aim of this study was to compare measures of limb function asymmetry during isometric and stretch-shortening cycle tasks and investigate agreement between the methods. Different between-limb effect sizes were observed between limbs dependent on test used, with different ranges of asymmetry observed. Agreement between methods was very poor. These results show the importance of assessing asymmetry in a condition that is as similar as possible to the task of interest

PLANTARFLEXOR FORCE PRODUCTION IN ISOMETRIC AND STRETCH-SHORTENING CYCLE TASKS

The ankle joint and its surrounding musculature are vital in sprinting, and recent work has developed a method to isolate the plantarflexor muscle group for analysis of its force-producing capabilities. The aim of this study was to compare values for plantarflexor force obtained using isometric dynamometry and an adapted force sledge in twenty healthy subjects. Results showed low relationships between isometric and dynamic strength measures, probably because the dynamic test utilises a stretch-shortening cycle. No relationship was observed between isometric strength and the sledge foot plate height during the task with a moderate relationship observed between dynamic strength and plate height. This highlights the important role the Achilles tendon plays in dynamic tasks

DO FLEXIBILITY SCREENING TESTS REFLECT JOINT RANGE OF MOTION DURING DYNAMIC TASKS?

The aim of this study was to investigate if ankle joint range of motion (ROM) during a functional screen test was related to ankle ROM and plantarflexor reactive strength index (RSI) during a dynamic task. The three measures were correlated for the dominant and non-dominant legs to identify what relationship, if any, existed between the three measures. Results showed that there was no relationship between the two measures of ROM, and ROM during the sledge task was positively related to plantarflexor RSI. This is in contrast to other work, possibly due to the isolation of the plantarflexor muscles in the sledge task. Interestingly, between-leg relationships were lower in the dynamic task, suggesting increased variability in the movement strategies used or underlying training differences

THE EFFECT OF INCREASED LOADING ON PLANTARFLEXOR FUNCTION DURING A STRETCH-SHORTENING CYCLE TASK

Increasing loading is one of the most common methods used to increase exercise intensity but it is important to consider the influence of the increase on subsequent force output, rate of force development and, in the case of plyometric exercise, contact time. The aim of this study was to investigate the effect of altering loading on plantarflexor force and force production during a fast stretch-shortening cycle task. Results showed load increased force production and contact time and decreased flight time and reactive strength index. These results have important implications for practitioners in determining the optimal method of increasing training intensity in order to avoid negative training effects

Using Instructional Logs to Study Mathematics Curriculum and Teaching in the Early Grades

In this article we describe the mathematics curriculum and teaching practices in a purposive sample of high‐poverty elementary schools working with 3 of the most widely disseminated comprehensive school reform programs in the United States. Data from 19,999 instructional logs completed by 509 first‐, third‐, and fourth‐grade teachers in 53 schools showed that the mathematics taught in these schools was conventional despite a focus in the schools on instructional improvement. The typical lesson focused on number concepts and operations, had students working mostly with whole numbers (rather than other rational numbers), and involved direct teaching or review and practice of routine skills. However, there was wide variation in content coverage and teaching practice within and among schools, with variability among teachers in the same school being far greater than variability among teachers across schools. The results provide an initial view of the state of mathematics education in a sample of schools engaged in comprehensive school reform and suggest future lines for research

PRELIMINARY ANALYSIS OF SLEDGE REACTION FORCES DURING CYCLICAL LOADING OF THE TRICEPS SURAE

To date, no methodology exists that can measure Achilles tendon stiffness in a controlled dynamic situation while simultaneously investigating tendon and joint stiffness interactions. Stiffness refers to the ratio between force and elongation, and the aim of this preliminary study was to establish an analysis protocol for sledge reaction forces during cyclical loading of the triceps surae. Results indicated the magnitude of forces was just under 50% of body weight, which was expected. Removal of the first two trials from analysis reduced standard deviation and 95% confidence interval of plantarflexor force, contact time and contact time-flight time ratio, suggesting this protocol is suitable to ensure data gathered is repeatable and consistent. Future work using inverse dynamics and ultrasound shall provide information on Achilles tendon loading and stiffness

CATHEDRAL: A Fast and Effective Algorithm to Predict Folds and Domain Boundaries from Multidomain Protein Structures

We present CATHEDRAL, an iterative protocol for determining the location of previously observed protein folds in novel multidomain protein structures. CATHEDRAL builds on the features of a fast secondary-structure–based method (using graph theory) to locate known folds within a multidomain context and a residue-based, double-dynamic programming algorithm, which is used to align members of the target fold groups against the query protein structure to identify the closest relative and assign domain boundaries. To increase the fidelity of the assignments, a support vector machine is used to provide an optimal scoring scheme. Once a domain is verified, it is excised, and the search protocol is repeated in an iterative fashion until all recognisable domains have been identified. We have performed an initial benchmark of CATHEDRAL against other publicly available structure comparison methods using a consensus dataset of domains derived from the CATH and SCOP domain classifications. CATHEDRAL shows superior performance in fold recognition and alignment accuracy when compared with many equivalent methods. If a novel multidomain structure contains a known fold, CATHEDRAL will locate it in 90% of cases, with <1% false positives. For nearly 80% of assigned domains in a manually validated test set, the boundaries were correctly delineated within a tolerance of ten residues. For the remaining cases, previously classified domains were very remotely related to the query chain so that embellishments to the core of the fold caused significant differences in domain sizes and manual refinement of the boundaries was necessary. To put this performance in context, a well-established sequence method based on hidden Markov models was only able to detect 65% of domains, with 33% of the subsequent boundaries assigned within ten residues. Since, on average, 50% of newly determined protein structures contain more than one domain unit, and typically 90% or more of these domains are already classified in CATH, CATHEDRAL will considerably facilitate the automation of protein structure classification

AN EMG PROFILE OF LOWER LIMB MUSCLES DURING LINEAR GLIDE AND STANDING SHOT PUTTING

The purpose of this study was to provide a descriptive analysis of the phasic muscle activity of 8 lower limb muscles during performance of the shot put field event in track and field athletics. Six shot putters performed 3 standing and 3 full linear glide technique throws. Electromyography (EMG) of 8 lower limb muscles was recorded during the trials and the distance thrown was also measured. A comparison between standing and glide techniques are important from a coaching perspective. An increase in peak muscle activity of the Rectus Femoris was observed between the glide and standing throw, all other lower limb muscles showed no significant increases between the techniques. Results show significant increases between performances of standing and glide throws in female athletes however the mean differences were smaller in the male athletes