Bioinformatic Extraction of Functional Genetic Diversity from Heterogeneous Germplasm Collections for Crop Improvement

Efficient utilization of genetic variation in plant germplasm collections is impeded by large collection size, uneven characterization of traits, and unpredictable apportionment of allelic diversity among heterogeneous accessions. Distributing compact subsets of the complete collection that contain maximum allelic diversity at functional loci of interest could streamline conventional and precision breeding. Using heterogeneous population samples from Arabidopsis, Populus and sorghum, we show that genomewide single nucleotide polymorphism (SNP) data permits the capture of 3–78 fold more haplotypic diversity in subsets than geographic or environmental data, which are commonly used surrogate predictors of genetic diversity. Using a large genomewide SNP data set from landrace sorghum, we demonstrate three bioinformatic approaches to extract functional genetic diversity. First, in a “candidate gene” approach, we assembled subsets that maximized haplotypic diversity at 135 putative lignin biosynthetic loci, relevant to biomass breeding programs. Secondly, we applied a keyword search against the Gene Ontology to identify 1040 regulatory loci and assembled subsets capturing genomewide regulatory gene diversity, a general source of phenotypic variation. Third, we developed a machine-learning approach to rank semantic similarity between Gene Ontology term definitions and the textual content of scientific publications on crop adaptation to climate, a complex breeding objective. We identified 505 sorghum loci whose defined function is semantically-related to climate adaptation concepts. The assembled subsets could be used to address climatic pressures on sorghum production. To face impending agricultural challenges and foster rapid extraction and use of novel genetic diversity resident in heterogeneous germplasm collections, whole genome resequencing efforts should be prioritized

Glueball mass measurements from improved staggered fermion simulations

We present the first 2+1 flavour spectrum measurements of glueball states using high statistics simulations with improved staggered fermions. We find a spectrum consistent with quenched measurements of scalar, pseudoscalar andtensor glueball states. The measurements were made using 5000 configurations at a lattice spacing of 0.123 fm and pion mass of 280 MeV and 3000 configurations at 0.092 fm with a pion mass of 360 MeV. We see some evidence of coupling to 2 pion states. We compare our results with the experimental glueball candidate spectrum as well as quenched glueball estimates.Comment: 22 pages, 19 figures and 8 tables, minor additions on mixing post-refere

The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry

Retrospective Study of Midazolam Protocol for Prehospital Behavioral Emergencies

Introduction: Agitated patients in the prehospital setting pose challenges for both patient care and emergency medical services (EMS) provider safety. Midazolam is frequently used to control agitation in the emergency department setting; however, limited data exist in the prehospital setting. We describe our experience treating patients with midazolam for behavioral emergencies in a large urban EMS system. We hypothesized that using midazolam for acute agitation leads to improved clinical conditions without causing significant clinical deterioration.Methods: We performed a retrospective review of EMS patient care reports following implementation of a behavioral emergencies protocol in a large urban EMS system from February 2014–June 2016. For acute agitation, paramedics administered midazolam 1 milligram (mg) intravenous (IV), 5 mg intramuscular (IM), or 5 mg intranasal (IN). Results were analyzed using descriptive statistics, Levene’s test for assessing variance among study groups, and t-test to evaluate effectiveness based on route.Results: In total, midazolam was administered 294 times to 257 patients. Median age was 30 (interquartile range 24–42) years, and 66.5% were male. Doses administered were 1 mg (7.1%) and 5 mg (92.9%). Routes were IM (52.0%), IN (40.8%), and IV (7.1%). A second dose was administered to 37 patients. In the majority of administrations, midazolam improved the patient’s condition (73.5%) with infrequent adverse events (3.4%). There was no significant difference between the effectiveness of IM and IN midazolam (71.0% vs 75.4%; p = 0.24).Conclusion: A midazolam protocol for prehospital agitation was associated with reduced agitation and a low rate of adverse events

Organelle-Specific Single-Molecule Imaging of \u3cem\u3eα\u3c/em\u3e4\u3cem\u3eβ\u3c/em\u3e2 Nicotinic Receptors Reveals the Effect of Nicotine on Receptor Assembly and Cell-Surface Trafficking

Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure

Utilizing pHluorin-Tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins

Anti-tumour effects of a specific anti-ADAM17 antibody in an ovarian cancer model in vivo.

ADAM 17 (TNF-α converting enzyme, TACE) is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α. In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC(50) for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α (4-15-fold reductions, p<0.0001 for all three). Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours

Mammalian Cell-Derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly

3-D kinematic comparison of treadmill and overground running.

Studies investigating the mechanics of human movement are often conducted using the treadmill. The treadmill is an attractive device for the analysis of human locomotion. Studies comparing overground and treadmill running have analyzed discrete variables, however differences in excursion from footstrike to peak angle and range of motion during stance have yet to be examined. This study aimed to examine the 3-D kinematics of the lower extremities during overground and treadmill locomotion to determine the extent to which the two modalities differ. Twelve participants ran at 4.0m/s in both treadmill and overground conditions. 3-D angular kinematic parameters during the stance phase were collected using an eight camera motion analysis system. Hip, knee and ankle joint kinematics were quantified in the sagittal, coronal and transverse planes, then compared using paired t-tests. Of the parameters analyzed hip flexion at footstrike 12° hip range of motion 17°, peak hip flexion 12.7°, hip transverse plane range of motion 8° peak knee flexion 5° and peak ankle excursion range 6.6°, coronal plane ankle angle at toe-off 6.5° and peak ankle eversion 6.3° were found to be significantly different. These results lead to the conclusion that the mechanics of treadmill locomotion cannot be generalized to overground