Advances in Agrobacterium transformation and vector design result in high-frequency targeted gene insertion in maize.

CRISPR-Cas is a powerful DNA double-strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here, we report an efficient, selectable marker-free site-specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium-mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.

Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion.

Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.

Superior field performance of waxy corn engineered using CRISPR-Cas9.

We created waxy corn hybrids by CRISPR-Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.

Uniform Expression and Relatively Small Position Effects Characterize Sister Transformants in Maize and Soybean.

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

Spinal manipulation and exercise for low back pain in adolescents: study protocol for a randomized controlled trial.

BACKGROUND: Low back pain is among the most common and costly chronic health care conditions. Recent research has highlighted the common occurrence of non-specific low back pain in adolescents, with prevalence estimates similar to adults. While multiple clinical trials have examined the effectiveness of commonly used therapies for the management of low back pain in adults, few trials have addressed the condition in adolescents. The purpose of this paper is to describe the methodology of a randomized clinical trial examining the effectiveness of exercise with and without spinal manipulative therapy for chronic or recurrent low back pain in adolescents. METHODS/DESIGN: This study is a randomized controlled trial comparing twelve weeks of exercise therapy combined with spinal manipulation to exercise therapy alone. Beginning in March 2010, a total of 184 participants, ages 12 to 18, with chronic or recurrent low back pain are enrolled across two sites. The primary outcome is self-reported low back pain intensity. Other outcomes include disability, quality of life, improvement, satisfaction, activity level, low back strength, endurance, and motion. Qualitative interviews are conducted to evaluate participants' perceptions of treatment. DISCUSSION: This is the first randomized clinical trial assessing the effectiveness of combining spinal manipulative therapy with exercise for adolescents with low back pain. The results of this study will provide important evidence on the role of these conservative treatments for the management of low back pain in adolescents. TRIAL REGISTRATION: (ClinicalTrials.gov NCT01096628).

Development and psychometric evaluation of an evidence-based practice questionnaire for a chiropractic curriculum.

OBJECTIVE: The purposes of this study were to describe the questionnaire development process for evaluating elements of an evidence-based practice (EBP) curriculum in a chiropractic program and to report on initial reliability and validity testing for the EBP knowledge examination component of the questionnaire. METHODS: The EBP knowledge test was evaluated with students enrolled in a doctor of chiropractic program in the University of Western States. The initial version was tested with a sample of 374 and a revised version with a sample of 196 students. Item performance and reliability were assessed using item difficulty, item discrimination, and internal consistency. An expert panel assessed face and content validity. RESULTS: The first version of the knowledge examination demonstrated a low internal consistency (Kuder-Richardson 20 = 0.55), and a few items had poor item difficulty and discrimination. This resulted in an expansion in the number of items from 20 to 40, as well as a revision of the poorly performing items from the initial version. The Kuder-Richardson 20 of the second version was 0.68; 32 items had item difficulties of between 0.20 and 0.80, and 26 items had item discrimination values of 0.20 or greater. CONCLUSIONS: A questionnaire for evaluating a revised EBP-integrated curriculum was developed and evaluated. Psychometric testing of the EBP knowledge component provided some initial evidence for acceptable reliability and validity.

Physical examination and self-reported pain outcomes from a randomized trial on chronic cervicogenic headache.

OBJECTIVE: Objective clinical measures for use as surrogate markers of cervicogenic headache (CGH) pain have not been established. In this analysis, we investigate relationships between objective physical examination (PE) measures with self-reported CGH outcomes. METHODS: This is an exploratory analysis of data generated by attention control PE from an open-label randomized clinical trial. Of 80 subjects, 40 were randomized to 8 treatments (spinal manipulative therapy or light massage control) and 8 PE over 8 weeks. The remaining subjects received no PE. Physical examination included motion palpation of the cervical and upper thoracic regions, active cervical range of motion (ROM) and associated pain, and algometric pain threshold evaluated over articular pillars. Self-reported outcomes included CGH and neck pain and disability, number of CGH headaches, and related disability days. Associations between PE and self-reported outcomes were evaluated using generalized linear models, adjusting for sociodemographic differences and study group. RESULTS: At baseline, number of CGH and disability days were strongly associated with cervical active ROM (P < .001 to .037). Neck pain and disability were strongly associated with ROM-elicited pain (P < .001 to .035) but not later in the study. After the final treatment, pain thresholds were strongly associated with week 12 neck pain and disability and CGH disability and disability days (P < or = .001 to .048). CONCLUSIONS: Cervical ROM was most associated with the baseline headache experience. However, 4 weeks after treatment, algometric pain thresholds were most associated. No one PE measure remained associated with the self-reported headache outcomes over time.

Pulmonary edema induced by cerebral hypoxic insult in a canine model.

INTRODUCTION: The mechanisms of noncardiogenic pulmonary edema syndromes, such as neurogenic and high-altitude pulmonary edema, remain unclear even after years of study. Previous attempts to develop an animal model for these illnesses have used increased intracranial pressure or whole-body hypoxia. We hypothesized that a cerebral insult induced with a venous hypoxic blood infusion to the brain would trigger neurogenic pulmonary edema in a canine model. METHODS: We measured indices of pulmonary edema, hemodynamics, norepinephrine (NE), and epinephrine values in anesthetized adult Walker hounds in which the brain was perfused for 2 h with either venous blood (venous perfused brain, VPB) or arterial blood (arterial perfused brain, APB) while maintaining normoxic pulmonary and systemic circulations. Normal cerebral perfusion was then reinstated for an additional 2-h period before euthanasia. RESULTS: VPB animals showed a greater fall in arterial PO2 and SaO2 and higher peak plasma NE compared to APB. On necropsy, VPB animals had greater lungwet-to-dry weight ratios compared to APB. Histological analyses revealed areas of marked alveolar infiltration of neutrophils and macrophages, acute hemorrhage, congestion, and alveolar edema in the VPB animals. DISCUSSION: This study supports the hypothesis that a cerebral insult from venous hypoxic blood can induce pulmonary edema. This method yields a promising approach to the study of noncardiogenic pulmonary edema syndromes.

Parecoxib: a shift in pain management?

Parecoxib (Dynastat) is a parenteral cyclooxygenase-2 inhibitor available in Europe. Clinical trials have reported a benefit in reducing pain following oral, orthopedic, gynecologic and cardiac surgeries. The overall efficacy was dose-related and similar to ketorolac (Toradol). Several trials reported an opioid-sparing effect with parecoxib. No trials have reported significantly fewer opioid-related gastrointestinal side effects (e.g., nausea, vomiting, constipation and intestinal ileus) when opioids were administered with parecoxib versus placebo. Gastroduodenal ulcers, gastric ulcers and duodenal ulcers or erosions were less common with parecoxib than ketorolac. Parecoxib does not affect platelet aggregation, interfere with the antiplatelet affect of aspirin, affect prothrombin and partial thromboplastin time or platelet counts when administered with heparin.