Targeted genome modifications in soybean with CRISPR/Cas9

Background: The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci. Results: Targeted DNA mutations were detected in 95% of 88 hairy-root transgenic events analyzed. Bi-allelic mutations were detected in events transformed with eight of the nine targeting vectors. Small deletions were the most common type of mutation produced, although SNPs and short insertions were also observed. Homoeologous genes were successfully targeted singly and together, demonstrating that CRISPR/Cas9 can both selectively, and generally, target members of gene families. Somatic embryo cultures were also modified to enable the production of plants with heritable mutations, with the frequency of DNA modifications increasing with culture time. A novel cloning strategy and vector system based on In-Fusion (R) cloning was developed to simplify the production of CRISPR/Cas9 targeting vectors, which should be applicable for targeting any gene in any organism. Conclusions: The CRISPR/Cas9 is a simple, efficient, and highly specific genome editing tool in soybean. Although some vectors are more efficient than others, it is possible to edit duplicated genes relatively easily. The vectors and methods developed here will be useful for the application of CRISPR/Cas9 to soybean and other plant species

Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples

Circadian Oscillations of Protein-Coding and Regulatory RNAs in a Highly Dynamic Mammalian Liver Epigenome

SummaryIn the mouse liver, circadian transcriptional rhythms are necessary for metabolic homeostasis. Whether dynamic epigenomic modifications are associated with transcript oscillations has not been systematically investigated. We found that several antisense RNA, lincRNA, and microRNA transcripts also showed circadian oscillations in adult mouse livers. Robust transcript oscillations often correlated with rhythmic histone modifications in promoters, gene bodies, or enhancers, although promoter DNA methylation levels were relatively stable. Such integrative analyses identified oscillating expression of an antisense transcript (asPer2) to the gene encoding the circadian oscillator component Per2. Robust transcript oscillations often accompanied rhythms in multiple histone modifications and recruitment of multiple chromatin-associated clock components. Coupling of cycling histone modifications with nearby oscillating transcripts thus established a temporal relationship between enhancers, genes, and transcripts on a genome-wide scale in a mammalian liver. The results offer a framework for understanding the dynamics of metabolism, circadian clock, and chromatin modifications involved in metabolic homeostasis

Exploring the Law of Detrital Zircon: LA-ICP-MS and CA-TIMS Geochronology of Jurassic Forearc Strata, Cook Inlet, Alaska, USA

Uranium-lead (U-Pb) geochronology studies commonly employ the law of detrital zircon: A sedimentary rock cannot be older than its youngest zircon. This premise permits maximum depositional ages (MDAs) to be applied in chronostratigraphy, but geochronologic dates are complicated by uncertainty. We conducted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and chemical abrasion-thermal ionization mass spectrometry (CA-TIMS) of detrital zircon in forearc strata of southern Alaska (USA) to assess the accuracy of several MDA approaches. Six samples from Middle–Upper Jurassic units are generally replete with youthful zircon and underwent three rounds of analysis: (1) LA-ICP-MS of ∼115 grains, with one date per zircon; (2) LA-ICP-MS of the ∼15 youngest grains identified in round 1, acquiring two additional dates per zircon; and (3) CA-TIMS of the ∼5 youngest grains identified by LA-ICP-MS. The youngest single-grain LA-ICP-MS dates are all younger than—and rarely overlap at 2σ uncertainty with—the CA-TIMS MDAs. The youngest kernel density estimation modes are typically several million years older than the CA-TIMS MDAs. Weighted means of round 1 dates that define the youngest statistical populations yield the best coincidence with CA-TIMS MDAs. CA-TIMS dating of the youngest zircon identified by LA-ICP-MS is indispensable for critical MDA applications, eliminating laser-induced matrix effects, mitigating and evaluating Pb loss, and resolving complexities of interpreting lower-precision, normally distributed LA-ICP-MS dates. Finally, numerous CA-TIMS MDAs in this study are younger than Bathonian(?)–Callovian and Oxfordian faunal correlations suggest, highlighting the need for additional radioisotopic constraints—including CA-TIMS MDAs—for the Middle–Late Jurassic geologic time scale

Changes in Drop-Jump Landing Biomechanics During Prolonged Intermittent Exercise

Background: As injury rates rise in the later stages of sporting activities, a better understanding of lower extremity biomechanics in the later phases of gamelike situations may improve training and injury prevention programs.Hypothesis: Lower extremity biomechanics of a drop-jump task (extracted from a principal components analysis) would reveal factors associated with risk of anterior cruciate ligament injury during a 90-minute individualized intermittent exercise protocol (IEP) and for 1 hour following the IEP.Study Design: Controlled laboratory study.Level of Evidence: Level 4.Methods: Fifty-nine athletes (29 women, 30 men) completed 3 sessions. The first session assessed fitness for an IEP designed to simulate the demands of a soccer match. An experimental session assessed drop-jump biomechanics, after a dynamic warm-up, every 15 minutes during the 90-minute IEP, and for 1 hour following the IEP. A control session with no exercise assessed drop-jump performance at the same intervals.Results: Two biomechanical factors early in the first half (hip flexion at initial contact and hip loading; ankle loading and knee shear force) decreased at the end of the IEP and into the 60-minute recovery period, while a third factor (knee loading) decreased only during the recovery period (P = 0.05).Conclusion: The individualized sport-specific IEP may have more subtle effects on landing biomechanics when compared with short-term, exhaustive fatigue protocols.Clinical Relevance: Potentially injurious landing biomechanics may not occur until the later stages of soccer activity

Changes in Fatigue, Multiplanar Knee Laxity, and Landing Biomechanics During Intermittent Exercise

Context: Knee laxity increases during exercise. However, no one, to our knowledge, has examined whether these increases contribute to higher-risk landing biomechanics during prolonged, fatiguing exercise.Objectives: To examine associations between changes in fatigue (measured as sprint time [SPTIME]), multiplanar knee laxity (anterior-posterior [APLAX], varus-valgus [VVLAX] knee laxity, and internal-external rotation [IERLAX]) knee laxity and landing biomechanics during prolonged, intermittent exercise.Design: Descriptive laboratory study.Setting: Laboratory and gymnasium.Patients or Other Participants: A total of 30 male (age = 20.3 ± 2.0 years, height = 1.79 ± 0.05 m, mass = 75.2 ± 7.2 kg) and 29 female (age = 20.5 ± 2.3 years, height = 1.67 ± 0.08 m, mass = 61.8 ± 9.0 kg) competitive athletes.Intervention(s): A 90-minute intermittent exercise protocol (IEP) designed to simulate the physiologic and biomechanical demands of a soccer match.Main Outcome Measure(s): We measured SPTIME, APLAX, and landing biomechanics before and after warm-up, every 15 minutes during the IEP, and every 15 minutes for 1 hour after the IEP. We measured VVLAX and IERLAX before and after the warm-up, at 45 and 90 minutes during the IEP, and at 30 minutes after the IEP. We used hierarchical linear modeling to examine associations between exercise-related changes in SPTIME and knee laxity with exercise-related changes in landing biomechanics while controlling for initial (before warm-up) knee laxity.Results: We found that SPTIME had a more global effect on landing biomechanics in women than in men, resulting in a more upright landing and a reduction in landing forces and out-of-plane motions about the knee. As APLAX increased with exercise, women increased their knee internal-rotation motion (P = .02), and men increased their hip-flexion motion and energy-absorption (P = .006) and knee-extensor loads (P = .04). As VVLAX and IERLAX increased, women went through greater knee-valgus motion and dorsiflexion and absorbed more energy at the knee (P = .05), whereas men were positioned in greater hip external and knee internal rotation and knee valgus throughout the landing (P = .03). The observed fatigue- and laxity-related changes in landing biomechanics during exercise often depended on initial knee laxity.Conclusions: Both exercise-related changes in fatigue and knee laxity were associated with higher-risk landing biomechanics during prolonged exercise. These relationships were more pronounced in participants with greater initial knee laxity

NLS cycle 1 and NLS 2 base heating technical notes. Appendix 3: Preliminary cycle 1 NLS base heating environments. Cycle 1 NLS base heating environments. NLS 2 650K STME base heating environments

A preliminary analysis of National Launch System ascent plume induced base heating environments has been completed to support the Induced Environments Panel's objective to assist in maturing the NLS vehicle (1.5 stage and heavy launch lift vehicle) design. Environments during ascent have been determined from this analysis for a few selected locations on the engine nozzles and base heat shield for both vehicles. The environments reflect early summer 1991 configurations and performance data and conservative methodology. A more complete and thorough analysis is under way to update these environments for the cycle 1 review in January 1992

Influence of Lean Body Mass and Strength on Landing Energetics

Purpose: Less lean body mass may limit one’s ability to produce adequate muscle forces to safely control landing from a jump, thus increasing the risk for injury. The primary objective of this study was to determine the effect of lower extremity lean mass (LELM) and eccentric muscle strength on lower extremity energy absorption (EA) during a drop jump landing.Methods: Seventy athletic subjects (35 men and 35 women) were measured for LELM with dual-energy x-ray absorptiometry, maximal eccentric strength of the quadriceps (QuadECC) and hamstrings (HamECC), and lower extremity joint energetics during the initial landing of a drop jump. A mediation analysis examined the extent to which LELM predicted EA at each lower extremity joint (EAHIP, EAKNEE, and EAANK) and subsequently whether these relationships were mediated by each subject’s maximal eccentric strength capabilities.Results: LELM was a significant predictor of EAKNEE (R2 = 0.22, P < 0.01) in females but not in males (R2 = 0.03, P = 0.16). In females, QuadECC was a significant mediator of the effect of LELM on EA at the knee (ab = 179.72, 95% confidence interval [CI] = 10.43–423.42) and ankle (ab = 1.71, 95% CI = [0.16, 3.94]), whereas HamECC was a significant mediator of the relationship between LELM and EAHIP (ab = 4.89, 95% CI = 2.05–8.40). No significant relationships were observed in males.Conclusions: LELM was a significant factor in energetic capabilities for females but not males. For females, this relationship was evident secondary to the stronger underlying relationship between maximal strength and EA. Thus, the maximal eccentric strength capabilities may be a more important determinant of energetic behaviors compared with the available quantity of lean mass alone. More work is needed to investigate these relationships and to reveal the underlying sex-specific mechanisms that determine EA capabilities