Production of [11C]cyanide for the synthesis of indole-3-[1-11C]acetic acid and PET imaging of auxin transport in living plants: Production of [11C]cyanide for the synthesis of indole-3-[1-11C]acetic acid and PET imaging of auxin transport in living plants

Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants

Parents, children and the porous boundaries of the sexual family in law and popular culture

This article focuses on a perceived ideological overlap between popular cultural and judicial treatments of sex and conjugality that contributes to a discursive construction of parenthood and parenting. The author perceives that in both legal and popular cultural texts, there is a sense in which notions of ‘natural’ childhood are discursively constituted as being put at risk by those who reproduce outside of dominant sexual norms, and that signs of normative sexuality (typically in the form of heterosexual coupling) may be treated as a sign of safety. These ideas are rooted in ancient associations between fertility, sexuality and femininity that can also be traced in the historical development of the English language. With the help of commentators such as Martha Fineman, the article situates parents and children within a discourse of family which prioritises conjugality, with consequences for the ways in which the internal and external boundaries of families are delineated

Pasture Conditions at the Initiation of Grazing to Optimize Forage Productivity: A Progress Report

To determine environmental, soil, and sward effects at the initiation of cattle grazing in the spring on seasonal (forage accumulated during the grazing season) and cumulative (seasonal + initial forage mass) forage accumulation (FA), 15 commercial cow-calf producers from southern Iowa were selected by historical initial grazing date. At grazing initiation, twelve .25-m2 samples were hand-clipped from each pasture and sward heights (SH) measured with a falling plane meter (4.8 kg/m2) to determine initial forage mass. At each location, soil temperature and load bearing capacity (LBC) were measured and a soil sample was collected to measure pH and moisture, P, and K concentrations. Cumulative degree-days (base=3.85°C) and precipitation at grazing initiation were calculated from NOAA records. At the beginning of each month, at least three grazing exclosures were placed on each grazed pasture to determine monthly FA. SH in each exclosure was recorded, and a .25-m2 forage sample was hand-clipped proximate to each exclosure. At the end of each month, SH was recorded and .25-m2 hand-clipped forage samples from inside exclosures were obtained. In linear regressions, cumulative and seasonal SH increased with greater soil P (r2=.5049 and .5417), soil K (r2=.4675 and .4397), and initial forage mass (r2=.1984 and .2801). Seasonal SH increased with earlier initial grazing dates (r2=.1996) and less accumulated degree-days (r2=.2364). Cumulative and seasonal FA increased with earlier initial grazing dates (r2=.2106 and .3744), lower soil temperatures (r2=.2617 and.2874), and greater soil P (r2=.3489 and .2598). Cumulative FA increased with greater soil K (r2=.4675). In quadratic regressions, cumulative and seasonal SH were correlated to soil P (r2=.6310 and .5310) and soil K (r2=.5095 and.4401). Cumulative and seasonal FA were correlated to degree days (r2=.3630 and.4013) and initial grazing date (r2=.3425 and .4088). Cumulative FA was correlated to soil P (r2=.3539), and seasonal FA was correlated to soil moisture (r2=.3688)

Production and isolation of 72As from proton irradiation of enriched 72GeO2 for the development of targeted PET/MRI agents

Introduction Two current major research topics in nuclear medicine are in the development of long-lived positron-emitting nuclides for imaging tracers with long biological half-lives and in theranostics, imaging nuclides which have a chemically analogous therapy isotope. As shown in TABLE 1, the radioisotopes of arsenic (As) are well suited for both of these tasks with several imaging and therapy isotopes of a variety of biologically relevant half-lives accessible through the use of small medical cyclotrons. The five naturally abundant isotopes of germanium are both a boon and challenge for the medical nuclear chemist. They are beneficial in that they facilitate a wide array of producible radioarsenic isotopes. They are a challenge as monoisotopic radioarsenic production requires isotopically-enriched targets that are expensive and of limited availability. This makes it highly desirable that the germanium target material is reclaimed from arsenic isolation chemistry. One major factor which has limited the development of radioarsenic has been difficulties in its incorporation into biologically relevant targeting vectors. Previous studies have labeled antibodies and polymers through covalent bonding of arsenite (As(III)) with the sulfydryl group1,2,3. Recent work in our group has shown the facile synthesis and utility of superparamagnetic iron oxide nanoparticle- (SPION-)bound radioarsenic as a dual modality positron emission tomography (PET)/magnetic resonance imaging (MRI) agent4. Presently, we have built upon previous studies producing, isolating, and labeling untargeted SPION with radioarsenic4,5. We have incorp-rated the use of isotopically-enriched 72GeO2 for the production of radioisotopically pure 72As. The bulk of the 72GeO2 target material was re-claimed from the arsenic isolation chemical procedure for reuse in future irradiations. The 72As was used for ongoing development toward the synthesis of targeted, As-SPION-based, dual-modality PET/MRI agents. Material and Methods Targets of ~100 mg of isotopically-enriched 72GeO2 (96.6% 72Ge, 2.86% 73Ge, 0.35% 70Ge, 0.2% 74Ge, 0.01% 76Ge, Isoflex USA) were pressed into a niobium beam stop at 225 MPa, covered with a 25 µm HAVAR containment foil, attached to a water-cooling target port, and irradiated with 3 µA of 16.1 MeV protons for 2–3 hours using a GE PETtrace cyclotron. After irradiation, the target and beam stop were assembled into a PTFE dissolution apparatus, where the 72GeO2 target material was dissolved with the addition of 2 mL of 4 M NaOH and subsequent stirring. After dissolution was completed, the clear, colorless solution was transferred to a fritted glass column and the bulk 72GeO2 was reprecipitated by neutralizing the solution with the addition of 630 µL [HCl]conc, filtered, and rinsed with 1 mL [HCl]conc. To the combined 72As-containing filtrates, 100 µL 30% H2O2 was added to ensure that 72As was in the nonvolatile As(V) oxidation state. The ~3 mL solution was then evaporated at 115 ˚C while the vessel was purged with argon, followed by a second addition of 100 µL H2O2 after the volume was reduced to 1 mL. When the filtrate volume was ~0.3 mL, the vessel was removed from heat, allowed to cool with argon flow, and the arsenic reconstituted in 1 mL [HCl]conc and loaded onto a 1.5 mL bed volume Bio-Rad AG 1×8, 200–400 mesh anion exchange column preconditioned with 10 M HCl. The radioarsenic was eluted in 10 M HCl in the next ~10 mL, with 90% of the activity eluting in a 4 mL fraction. The column was then eluted with 5 mL 1 M HCl. The 72As-rich 10 M HCl fraction was reduced to As(III) with the addition of ~100 mg CuCl, and heating to 60 ˚C for 1 hour. The resulting AsCl3 was then extracted twice into 4 mL cyclohexane, which were combined and back extracted into 500 µL of water as As(OH)3. This solution of 72As in H2O was then used directly to label SPION and for subsequent experiments conjugating 72As-SPION with TRC105, an angiogenesis-marking monoclonal antibody (MAb) targeting endoglin/CD105. Several methods were initially attempted involving directly conjugating the surface-modified SPION to the MAb through a polyethylene glycol (PEG) linker. More recent studies have investigated the radioarsenic labeling of SPION encapsulated in hollow mesoporous silica nanoparticles (SPION@HMSN) and its subsequent conjugation to TRC105. Results and Conclusion Irradiation of pressed, isotopically-enriched 72GeO2 resulted in a production yield for 72As of 17 ± 2 mCi/(µA·hr·g) and for 71As of 0.37 ± 0.04 mCi/(µA·hr·g), which are 64 % and 33 %, of those predicted from literature6, respectively. However, these production yields are in agreement with those scaled from observed production yields using analagous natGeO2 targets. The end-of-bombardment 72As radionuclidic purity can be improved by minimizing the 72Ge(p,2n)71As reaction by degrading the beam energy. A 125 µm Nb containment foil would degrade impinging protons to 14.1 MeV and is predicted to reduce 71As yield by a factor of three, while only reducing 72As yield by 1 %6, improving end-of-bombardment radionuclidic purity from 98 % to greater than 99 %. Overall decay-corrected radiochemical yield of the 72As isolation procedure from 72GeO2 were 51 ± 2 % (n = 3) in agreement with those observed with natGeO2 57 ± 7 % (n = 14). The beam current was limited to 3 µA as higher cur-rents 4–5 µA exhibited inconsistent dissolution and reprecipitation steps, resulting in an overall yield of 44 ± 21 % (n = 6). Dissolution time also played an important role in overall yield with at least one hour necessary to minimize losses in these first two steps. The separation procedure effectively removed all radiochemical contaminants and resulted in 72As(OH)3 isolated in a small volume, pH~4.5 water solution. Over the course of minutes to hours after back extraction, rapid auto-oxidation to 72AsO4H3 was observed. The bulk 72GeO2 target material, which was reclaimed from the isolation procedure, is being collected for future use. The synthesis of a targeted PET/MRI agent based on the functionalization of 72As-SPION has proved to be a difficult task. Experiments conjugating 72As-SPION to TRC105 through a PEG linker were unsuccessful, despite the investigation of a variety bioconjugation procedures. Current work is investigating the use of SPION@HMSN, which have a similar affinity for 72As as unencapsulated SPION. This new class of 72As-labeled SPION@HMSN has a hollow cavity for potential anti-cancer drug loading, as well as the mesoporous silica surface, which may facilitate the efficient conjugation of TRC105 using a well-developed bioconjugation technique. In summary, radioarsenic holds potential in the field of diagnostic and therapeutic nuclear medicine. However, this potential remains locked behind challenges related to its production and useful in vivo targeting. The present work strives to address several of these challenges through the use of enriched 72GeO2 target material, a chemical isolation procedure that reclaims the bulk of the target material, and the investigation of new targeted nanoparticle-based PET/MRI agents

Envelope Protection for In-Flight Ice Contamination

Fatal loss-of-control (LOC) accidents have been directly related to in-flight airframe icing. The prototype system presented in this paper directly addresses the need for real-time onboard envelope protection in icing conditions. The combinations of a-priori information and realtime aerodynamic estimations are shown to provide sufficient input for determining safe limits of the flight envelope during in-flight icing encounters. The Icing Contamination Envelope Protection (ICEPro) system has been designed and implemented to identify degradations in airplane performance and flying qualities resulting from ice contamination and provide safe flight-envelope cues to the pilot. Components of ICEPro are described and results from preliminary tests are presented

Integration of Year-round Forage Management Systems for Spring-calving and Fall-calving Beef Cows (A Progress Report)

Animal production, hay production and feeding, winter forage composition changes, and summer pasture yields and nutrient composition of a year-round grazing system for spring-calving and fall-calving cows were compared to those of a conventional, minimal land system. Cows in the year-round and minimal land systems grazed forage from smooth bromegrassorchardgrass-birdsfoot trefoil (SB-O-T) pastures at 1.67 and 3.33 acres, respectively, per cow in the summer. During the summer, SB-O-T pastures in the year-round grazing system also were grazed by stockers at 1.67 stockers per acre, and spring-calving and fall-calving cows grazed smooth bromegrass–red clover (SB-RC) and endophyte-free tall fescue–red clover (TF-RC) at 2.5 acres per cow for approximately 45 days in midsummer. In the year-round grazing system, spring-calving cows grazed corn crop residues at 2.5 acres per cow and stockpiled SB-RC pastures at 2.5 acres per cow; fallcalving cows grazed stockpiled TF-RC pastures at 2.5 acres per cow during winter. In the minimal land system, in winter, cows were maintained in a drylot on first-cutting hay harvested from 62.5–75% of the pasture acres during summer. Hay was fed to maintain a body condition score of 5 on a 9-point scale for springcalving cows in both systems and a body condition score of 3 for fall-calving cows in the year-round system. Over 3 years, mean body weights of fall-calving cows in the year-round system did not differ from the body weights of spring-calving cows in either system, but fall-calving cows had higher (P \u3c .05) body condition scores compared to spring-calving cows in either system. There were no differences among all groups of cows in body condition score changes over the winter grazing season (P \u3e .05). During the summer grazing season, fall-calving cows in the year- round system and springcalving cows in the minimal land system gained more body condition and more weight (P \u3c .05) than springcalving cows in the year-round grazing system. Fall calves in the year-round system had higher birth weights, lower weaning weights, and lower average preweaning daily gains compared to either group of spring calves (P \u3c .05). However, there were no significant differences for birth weights, weaning weights, or average pre-weaning daily gains between spring calves in either system over the 3-year experiment (P \u3e .05). The amount of total growing animal production (calves and stockers) per acre for each system did not differ in any year (P \u3e .05). Over the 3-year experiment, 1.9 ton more hay was fed per cow and 1 ton more hay was fed per cow–calf pair in the minimal land system compared to the year-round grazing system (P \u3c .05)

Evaluation of Year-round Forage Management Systems for Spring- and Fall-Calving Beef Cows (A Progress Report)

A year-round grazing system for spring- and fall-calving cows was developed to compare animal production and performance, hay production and feeding, winter forage composition changes, and summer pasture yield and nutrient composition to that from a conventional, or minimal land system. Systems compared forage from smooth bromegrass-orchardgrass-birdsfoot trefoil pastures for both systems in the summer and corn crop residues and stockpiled grass-legume pastures for the year-round system to drylot hay feeding during winter for the minimal land system. The year-round grazing system utilized 1.67 acres of smooth bromegrassorchardgrass- birdsfoot trefoil (SB-O-T) pasture per cow in the summer, compared with 3.33 acres of (SB-O-T) pasture per cow in the control (minimal land) system. In addition to SB-O-T pastures, the year-round grazing system utilized 2.5 acres of tall fescue-red clover (TFRC) and 2.5 acres of smooth bromegrass-red clover (SBRC) per cow for grazing in both mid-summer and winter for fall- and spring-calving cows, respectively. First-cutting hay was harvested from the TF-RC and SB-RC pastures, and regrowth was grazed for approximately 45 days in the summer. These pastures were then fertilized with 40 lbs N/acre and stockpiled for winter grazing. Also utilized during the winter for spring-calving cows in the year-round grazing system were corn crop residue (CCR) pastures at an allowance of 2.5 acres per cow. In the minimal land system, hay was harvested from three-fourths of the area in SB-O-T pastures and stored for feeding in a drylot through the winter. Summer grazing was managed with rotational stocking for both systems, and winter grazing of stockpiled forages and corn crop residues by year-round system cows was managed by strip-stocking. Hay was fed to maintain a body condition score of 5 on a 9 point scale for spring-calving cows in both systems. Hay was supplemented as needed to maintain a body condition score of 3 for fall-calving cows nursing calves through the winter. Although initial condition scores for cows in both systems were different at the initiation of grazing for both winter and summer, there were no significant differences (P \u3e .05) in overall condition score changes throughout both grazing seasons. In year 1, fall-calving cows in the year-round grazing system lost more (P \u3c .05) body weight during winter than spring-calving cows in either system. In year 2, there were no differences seen in weight changes over winter for any group of cows. Average daily gains of fall calves in the yearround system were 1.9 lbs/day compared with weight gains of 2.5 lbs/day for spring calves from both systems. Yearly growing animal production from pastures for both years did not differ between systems when weight gains of stockers that grazed summer pastures in the year-round grazing system were added to weight gains of suckling calves. Carcass characteristics for all calves finished in the feedlot for both systems were similar. There were no significant differences in hay production between systems for year 1; however, amounts of hay needed to maintain cows were 923, 1373, 4732 lbs dry matter/cow for year-round fall-calving, year-round spring-calving, and minimal land spring-calving cows, respectively. In year 2, hay production per acre in the minimal land system was greater (P \u3c .05) than for the year-round system, but the amounts of hay required per cow were 0, 0, and 4720 lbs dry matter/cow for yearround fall-calving, year-round spring-calving, and minimal land spring-calving cows, respectively

Geodetic Constraints on the 2014 M 6.0 South Napa Earthquake

On 24 August 2014, the M 6.0 South Napa earthquake shook much of the San Francisco Bay area, leading to significant damage in the Napa Valley. The earthquake occurred in the vicinity of the West Napa fault (122.313° W, 38.22° N, 11.3 km), a mapped structure located between the Rodger’s Creek and Green Valley faults, with nearly pure right‐lateral strike‐slip motion (strike 157°, dip 77°, rake –169°; http://comcat.cr.usgs.gov/earthquakes/eventpage/nc72282711#summary, last accessed December 2014) (Fig. 1). The West Napa fault previously experienced an M 5 strike‐slip event in 2000 but otherwise exhibited no previous definitive evidence of historic earthquake rupture (Rodgers et al., 2008; Wesling and Hanson, 2008). Evans et al. (2012) found slip rates of ∼9.5  mm/yr along the West Napa fault, with most slip rate models for the Bay area placing higher slip rates and greater earthquake potential on the Rodger’s Creek and Green Valley faults, respectively (e.g., Savage et al., 1999; d’Alessio et al., 2005; Funning et al., 2007)