Cereal transformation and gene editing can be a complex and costly undertaking. It is therefore important to validate and understand the performance of the components to achieve high rates of transformation and gene editing. Here, we have made a direct comparison of different CRISPR/Cas9 guide systems to target the genome in three cereal species. We show that the guide sequences driven by the same pol II promoter in rice, wheat and barley show large differences in editing efficiency. The differences seen were based on the way the guides were presented and factors outside of the guide sequence itself. While both the tRNA system and ribozyme system performed well in rice, their effectiveness varied in wheat and barley. Specifically, the tRNA system outperformed the ribozyme system, achieving higher rates of editing in stable transformed plants. Overall, high levels of editing are observed in all three species when strong expression of the SpCas9 is coupled with the CmYLCV promoter to drive a tRNA array of guide RNAs. Stable inheritance is also achievable in all three species when plants are sampled shortly after the tissue culture concludes. Overall, inheritance rates were above 85% in all three species, particularly when mutations are detected early after plants emerge from tissue culture.

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Oilseed rape (Brassica napus) is an important crop that is cultivated for the oil (mainly triacylglycerol; TAG) it produces in its seeds. TAG synthesis is controlled mainly by key enzymes in the Kennedy pathway, such as glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) but can also be produced from phosphoglycerides such as phosphatidylcholine (PC) by the activity of the enzyme phospholipid: diacylglycerol acyltransferase (PDAT). To evaluate the potential for these enzymes to alter oil yields or composition, we analysed transgenic B. napus lines which overexpressed GPAT, LPAT or PDAT using heterologous transgenes from Arabidopsis and Nasturtium and examined lipid profiles and changes in gene expression in these lines compared to WT. Distinct changes in PC and TAG abundance and spatial distribution in embryonic tissues were observed in some of the transgenic lines, together with altered expression of genes involved generally in acyl-lipid metabolism. Overall our results show that up-regulation of these key enzymes differentially affects lipid composition and distribution as well as lipid-associated gene expression, providing important information which could be used to improve crop properties by metabolic engineering.

OBJECTIVE:Little is understood about what contributes to perceived workload for those providing overnight coverage to hospitalized patients overnight, which limits the ability to modify these factors or to proactively identify appropriate staffing levels. The objective of this study is to understand the major contributors to perceived overnight cross-coverage workload.METHODS:Cross-covering advanced practice providers (APPs) in a large academic hospitalist group completed the National Aeronautics and Space Administration Task Load Index (NASA-TLX) at the end of each night shift. Other shift characteristics were collected, including patient load, assigned action items, watcher/unstable patients, newly admitted patients, number of units covered, total pages, peak pager density, rapid response team (RRT) activations, and intensive care unit (ICU) transfers.RESULTS:For 14 APP participants, who completed 271 post-shift surveys, the mean (SD) patient load was 49.9 (6.4) patients per night, and providers received a mean (SD) of 40.8 (13.7) total pages per shift. Mean (SD) NASA-TLX score was 35.1 (19.0). In multivariate modeling, total pages, action items, and any RRT or ICU transfer were associated with significant increases in the mean NASA-TLX score, with estimated effect sizes of 0.5, 0.8, and 14.3, respectively, per 1-unit increase in each shift characteristic. The greatest cumulative contributor to perceived workload was total number of pages, followed by the presence of any RRT activation/ICU transfer, with estimated effect sizes of 20.4 and 14.9, respectively.CONCLUSIONS:Total number of pages was the greatest contributor to perceived workload. This study suggests that quality improvement initiatives designed to improve pager communication may considerably improve provider-perceived workload.

Wheat is grown on more land than any other crop in the world. Current estimates suggest that yields will have to increase sixty percent by 2050 to meet the demand of an ever-increasing human population; however, recent wheat yield gains have lagged behind other major crops such as rice and maize. One of the reasons suggested for the lag in yield potential is the lack of a robust hybrid system to harness the potential yield gains associated with heterosis, also known as hybrid vigor. Here, we set out to identify candidate genes for a genic hybrid system in wheat and characterize their function in wheat using RNASeq on stamens and carpels undergoing meiosis. Twelve genes were identified as potentially playing a role in pollen viability. CalS5- and RPG1-like genes were identified as pre- and post-meiotic genes for further characterization and to determine their role in pollen viability. It appears that all three homoeologues of both CalS5 and RPG1 are functional in wheat as all three homoeologues need to be knocked out in order to cause male sterility. However, one functional homoeologue is sufficient to maintain male fertility in wheat.

OBJECTIVE:To evaluate the efficacy and safety of a fixed-dose gabapentin taper protocol for alcohol withdrawal in hospitalized patients.PATIENTS AND METHODS:We retrospectively identified patients admitted to the hospital from January 1, 2016, to April 30, 2018, for alcohol withdrawal syndrome. Based on the treatment that patients received, they were divided into the gabapentin, benzodiazepine, and combination treatment groups. The primary outcome was length of stay, defined as time from admission to either discharge or 36 hours with Clinical Institute Withdrawal Assessment (CIWA) score less than 10. Inverse probability of treatment weight was used to account for differences in baseline characteristics between groups.RESULTS:A total of 443 patients met criteria for inclusion (128, 253, and 62 patients in the gabapentin, benzodiazepine, and combination groups, respectively). Baseline characteristics were similar among all groups. The median gabapentin group length of stay was 4.0 hours shorter than the benzodiazepine group (P=.012). Maximum CIWA score was 2.2 points lower in the gabapentin group (P=.003). No statistical differences were noted among safety outcomes, including incidence of seizure, intensive care unit transfer, or delirium tremens. Results were not statistically altered by inverse probability of treatment weight analysis.CONCLUSION:A fixed-dose gabapentin taper protocol appears to be an effective and safe alternative to CIWA-driven benzodiazepines in patients hospitalized with alcohol withdrawal syndrome, though further research is necessary to define the potential subpopulations that benefit most.

Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1–2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.

Summary Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT. We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential. Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14C‐glycerol between phosphoglycerides. Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products.

The following method enables the rapid production of transgenic potato plants and microtubers for gene validation and expression, or promoter studies. The method is highly efficient, with reproducible transformation efficiencies of at least 50% to 60% with potato cv. Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment. Microtubers are produced in the absence of hormones, giving an in vitro gene testing system broadly analogous to the natural state. © 2018 by John Wiley & Sons, Inc.

Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens-mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc.

While many hospitalized patients have orders to fast in preparation for interventions, the extent to which these orders are necessary or adhere to evidence-based durations is unknown. In this study, we analyzed the length, indication, and associated outcomes of nil per os (NPO) orders for general medicine patients at an academic institution in the United States, and compared them to the best available evidence for recommended length of NPO. Of 924 NPO orders assessed, the indicated intervention was not performed for 183 (19.8%) orders, largely due to a change in plan (75/183, 41.0%) or scheduling barriers (43/183, 23.5%). When analyzed by indication, the median duration of NPO orders ranged from 8.3 hours for kidney ultrasound to 13.9 hours for upper endoscopy. For some indications, the literature suggested NPO orders may be unnecessary. Furthermore, in indications for which NPO was deemed necessary in the literature, the duration of most NPO orders was much longer than minimally required. These results suggest the need for establishing more robust practice guidelines or institutional protocols for NPO orders. Journal of Hospital Medicine 2017;12:36-39.

Objective: To evaluate the impact of screening stress testing for coronary artery disease in asymptomatic patients with diabetes in a community-based population.Patients and Methods: This observational study included 3146 patients from Olmsted County, Minnesota, with no history of coronary artery disease or cardiac symptoms in whom diabetes was newly diagnosed from January 1, 1992, through December 31, 2008. With combined all-cause mortality and myocardial infarction as the primary outcome, weighted Cox proportional hazards regression was performed with screening stress testing within 2 years of diabetes diagnosis as the time-dependent covariate. For descriptive analysis, participants were classified by their clinical experience during the first 2 years postdiagnosis as screened (asymptomatic, underwent stress test), unscreened (asymptomatic, no stress test), or symptomatic (experienced symptoms or event).Results: Among the screened and unscreened participants, 54% (1358 of 2538) were men; the mean (SD) age at diabetes diagnosis was 55 years (13.8 years), and 97% (2442 of 2520) had type 2 diabetes. In event-free survival analysis, 292 patients comprised the screened cohort and 2246 patients comprised the unscreened cohort. Death or myocardial infarction occurred in 454 patients (32 patients in the screened cohort and 422 in the unscreened cohort [5-year rate, 1.9% and 5.3%, respectively]) during median (interquartile range) follow-up of 9.1 years (5.3-12.5 years). Screening stress testing was associated with improved event-free survival (hazard ratio, 0.61; P=.004), independent of cardiac risk factors. However, while stress test results were abnormal in 47 of the 292 screened patients (16%), only 6 (2%) underwent coronary revascularization.Conclusion: Although screening cardiac stress testing in asymptomatic patients with diabetes in this community-based population was associated with improvement in long-term event-free survival, this result does not appear to occur by coronary revascularization alone. (C) 2016 Mayo Foundation for Medical Education and Research

OBJECTIVE:To evaluate the frequency and appropriateness of nil per os (nothing by mouth) (NPO) orders and determine the number of meals missed because of these orders among hospitalized patients.PATIENTS AND METHODS:We retrospectively analyzed inpatient NPO orders at an academic institution in the United States. The frequency and duration of NPO orders and the number of meals missed because of these orders were assessed for adult patients admitted to the hospital medicine services from January 1, 2013, through December 31, 2013, with a hospital stay of 2 or more and 30 or fewer days. Two blinded reviewers assessed if the order could be avoided or the period shortened for a random sample of NPO orders of 120 or more minutes' duration that were written for patients on the general medicine ward.RESULTS:A total of 3641 NPO orders were identified. At least one NPO order was placed in 46.6% of the admissions (2211 of 4743). The median duration of NPO orders was 12.8 hours (interquartile range, 9.2-17.3 hours), resulting in 2 (interquartile range, 1-4) missed meals. Of 1130 NPO orders reviewed, 263 (23.3%; 95% CI, 20.9%-25.8%) were deemed avoidable (κ statistic, 0.68), and 482 (42.7%) were unavoidable but led to more missed meals than needed. Taken together, patients could have had 44.8% of the meals (1085 of 2424; 95% CI, 42.8%-46.7%) missed due to NPO orders.CONCLUSION:Approximately half of the patients admitted to the hospital medicine services experienced a period of fasting. One in 4 NPO orders and nearly half of missed meals could have been avoided. Further study is warranted to assess the generalizability of our findings.

The comparative phenotypic analysis of mutants is often hampered by their diverse and poorly characterised genetic backgrounds. To overcome this problem, a suite of recombinant spring barley lines was developed for four starch biosynthesis genes in a common elite background. Rapid breeding progress was made by combining foreground and background selection with the screening of bulked families. A toolkit of perfect co-dominant PCR assays was developed for the four target genes, based on the causative single nucleotide polymorphisms underlying their starch phenotypes. These were used for foreground selection during backcrossing and selfing, and may be applied to bulks of up to ten plants. Screening bulks meant that large numbers of individuals with known family structure were rapidly assessed and that breeding effort was accurately targeted. These markers were also used for quality control during field multiplication and should be readily transferable to any crosses involving these four mutations. Background selection amongst BC1 progeny known to be heterozygous for the target starch alleles identified individuals which were relatively enriched for the recurrent parent across the rest of the genome. These were further advanced and true-breeding recombinants were selected which carry the target starch mutations in a largely recurrent parent background. The resulting set of BC2F5 pre-breeding lines should enable meaningful analysis of the starch phenotypes and facilitate their transfer into commercial breeding programmes.