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Summary 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.

Much attention has been given to the comedy of Mark Rylance’s Shakespeare performances and his interaction with the audience. Comedy and audience interaction informed the initial creation of many o...

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 knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an 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.

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.

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.

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.

This new design enabled an efficient layout of breakdown test devices for parametric and reliability testing. These reliability circuits consisted of interlocking combs of routing layers with varying widths and spaces that were representative of the design rules. These were accessed through multiplexer controlled pass gates. All addresses could be simultaneously enabled for stress biasing and addressed individually for failure detection. Once a breakdown was detected, as current leakage of the comb, each device could be addressed sequentially to find the failing structure. This was an improvement over previous designs which either grouped many devices in parallel, but could not electrically identify which device was failing, or only had a single device enabled, but suffered from poor pad efficiency. The grouping of these devices allows for simultaneous parallel stressing of each force and ground pad pair on the parametric testers. Electrical measurement showed that the same breakdown voltage values measured on this mux design were the same as standalone devices.

Author(s): Lamb, KD; Perring, AE; Samset, B; Peterson, D; Davis, S; Anderson, BE; Beyersdorf, A; Blake, DR; Campuzano-Jost, P; Corr, CA; Diskin, GS; Kondo, Y; Moteki, N; Nault, BA; Oh, J; Park, M; Pusede, SE; Simpson, IJ; Thornhill, KL; Wisthaler, A; Schwarz, JP | Abstract: East Asia is the strongest global source region for anthropogenic black carbon (BC), the most important light-absorbing aerosol contributing to direct radiative climate forcing. To provide extended observational constraints on regional BC distributions and impacts, in situ measurements of BC were obtained with a single particle soot photometer during the May/June 2016 Korean-United States Air Quality aircraft campaign (KORUS-AQ) in South Korea. Unique chemical tracer relationships were associated with BC sourced from different regions. The extent and variability in vertical BC mass burden for 48 profiles over a single site near Seoul were investigated using back trajectory and chemical tracer analysis. Meteorologically driven changes in transport influenced the relative importance of different source regions, impacting observed BC loadings at all altitudes. Internal mixing and size distributions of BC further demonstrated dependence on source region: BC attributed to China had a larger mass median diameter (180n±n13nnm) than BC attributed to South Korea (152n±n25nnm), and BC associated with long-range transport was less thickly coated (60n±n4nnm) than that sourced from South Korea (75n±n16nnm). The column BC direct radiative effect at the top of the atmosphere was estimated to be 1.0+0.90.5 W/m2, with average values for different meteorological periods varying by a factor of 2 due to changes in the BC vertical profile. During the campaign, BC sourced from South Korea (≤ 31%), China (22%), and Russia (14%) were the most significant single-region contributors to the column direct radiative effect.

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.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.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 (Por = .001 to .048).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.

We investigate the use of the Brace–Gatarek–Musiela (BGM) model in potential future exposure (PFE) calculations for various exotic interest rate products. Monte Carlo simulation is required to generate PFE scenarios. If the pricing model is also implemented using Monte Carlo simulation, PFE calculations are exceptionally demanding, involving nested Monte Carlo simulations. To reduce computation time, we look at two approximate pricing methods—a Longstaff-Schwartz regression-based approach, and a finite difference approach using a drift approximated BGM model. Numerical results are presented for several different exotic trade types. PFE results delivered by the two approximate methods are compared with results obtained by full resimulation, i.e. nested Monte Carlo. In the test cases we looked at, both approaches produced good results and were several orders of magnitude faster than full resimulation. However, care must be taken when using the regression-based approach. In particular, results can vary significantly depending on the order of the polynomial basis functions used in the regressions. Copyright © 2009 Wilmott Magazine Ltd

On 19 August 2005, more than nine-tenths of the members of both houses of Burundi's legislature capped a free electoral process by choosing Pierre Nkurunziza to serve as the country's next president. The implications of this event in this small, conflict-ridden state on the northeastern shore of Lake Tanganyika could reverberate positively throughout the Great Lakes region of central Africa and beyond. While Burundi's democratic reconstruction is far from complete, the election suggests that even the most violent and sorely divided societies can be restored, given sufficient political will and generous resources to support the process of negotiation and change.

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.

Negotiations to end Sudan's devastating civil war have repeatedly faltered, but among other developments, the re-emergence of civil society in both the north and south provides greater impetus for peace, as well as greater prospects for democracy and respect for human rights. Particularly important, but thus far relatively unnoticed, is the rise of a southern-led civil society movement in the north. Although Sudan's complex history and demographics provide an explanation for much of the conflict, in fact the conflict is essentially political, while it is this very complexity that could provide a basis for the resolution of the conflict. Some intriguing parallels with South Africa's recent experience underscore the role of civil society, and point to some options for the future.

(1998). Finding African solutions to African problems. The Washington Quarterly: Vol. 21, No. 3, pp. 149-158.

We have generated transgenic maize seed containing β-glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant β-glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant β-glucuronidase (GUS) was compared to native β-glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a Km of 0.20 mM and a Vmax of 0.29 mM MUG per hour, and the native enzyme had a Km and Vmax of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of β-glucuronidase, the Ki of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.

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.

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 Po 2 and S a O 2 and higher peak plasma NE compared to APB. On necropsy, VPB animals had greater lung wet-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.

Method A volume conductor model of a low-thoracic spinal cord with single and multiple epidurally-positioned cylindrical percutaneous leads was created using the finite element model tool ANSYS from which the electric field was calculated. The electric field results were then coupled with the NEURON simulator to determine the activated region of spinal cord DC and DR fibers [2]. DC and DR fiber models were adopted from double-cable axon model [3] with various fiber sizes (5.7–15 um diameter).

Tumors exhibit altered metabolic profiles compared to normal, non-transformed tissues. In the presence of oxygen, most differentiated cells convert glucose to ATP by oxidative phosphorylation (OX-PHOS), whereas tumor cells consume glucose and convert it to lactate, a phenomenon known as “aerobic glycolysis” or the “Warburg effect”. There is increased interest in targeting glycolysis as a therapeutic strategy in oncology, and one of the key enzymes in this pathway is lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate in the last step of glycolysis. A small molecule LDHA inhibitor that was identified by high throughput screening was optimized to generate GNE-140, which has a biochemical EC50 of 0.005 μM and inhibits MiaPaCa-2 pancreatic cell proliferation with an EC50 of 0.25 μM. We show that inhibition of LDHA reduces carbon flux from pyruvate to lactate, impairs glycolysis, and leads to global effects in cell metabolism. Cells cease proliferation within 24 hours of LDHA inhibition, yet only undergo cell death following sustained pathway inhibition. Finally, we show that pancreatic cell lines that are resistant to LDHA inhibition can be re-sensitized to LDHA inhibition by inhibiting OX-PHOS. In conclusion, we describe a potent cell active LDHA inhibitor that modulates glycolysis, induces cell death, and that can be used to help evaluate the importance of LDHA in tumor growth. Citation Format: Thomas O'Brien, Hans Purkey, Anna Hitz, Dave Peterson, Aaron Boudreau, Christopher Delnagro, Mandy Kwong, Rebecca Hong, Min Gao, Jodi Pang, Alex Vanderbilt, Simon Williams, Laurent Salphati, Deepak Sampath, Georgia Hatzivassiliou, Marie Evangelista. Inhibiting glycolysis with an LDHA inhibitor: A new solution to an old problem. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2014-964

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).