Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging.

Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research.

Evaluation of the Efficacy of Commercial Disinfectants Against Fusarium oxysporum f. sp. cubense Race 1 and Tropical Race 4 Propagules.

Panama disease caused by Fusarium oxysporum f. sp. cubense has devastated banana production worldwide. This work aimed to determine effective disinfectants against two races of F. oxysporum f. sp. cubense, race 1 and tropical race 4 (TR4), for implementation with on-farm biosecurity procedures against this disease following the outbreak of TR4 in North Queensland in 2015. A total of 32 commercial disinfectants were screened and their activity was assessed after ≤30 s, 5 min, 30 min, and 24 h of contact with an F. oxysporum f. sp. cubense suspension containing 105 chlamydospores/ml without and with soil added (0.05 g/ml). Of the disinfectants tested, the quaternary ammonium compounds containing ≥10% active ingredient were found to be the most effective against both F. oxysporum f. sp. cubense races. These products, when used at a 1:100 dilution, completely inhibited the survival of all F. oxysporum f. sp. cubense propagules across all the contact times regardless of the absence or presence of soil. The bioflavonoid product EvoTech 213 and bleach (10% sodium hypochlorite) used at a 1:10 dilution also eliminated all F. oxysporum f. sp. cubense propagules across all the contact times. None of the detergent-based or miscellaneous products tested were completely effective against both F. oxysporum f. sp. cubense races even used at a 1:10 dilution. Soil decreases the efficacy of disinfectants and therefore must be removed from contaminated items before treatments are applied.

Short communication: Lysine retained among 2 lipid-coated lysine products after exposure to alfalfa or corn silage with different amounts of acidity.

We conducted 2 experiments to determine lysine loss from 2 lipid-coated lysine products after mixing with silage. In our first experiment, we mixed 2 lipid-coated lysine products, crystalline lysine or crystalline lysine and amounts of lipid identical to amounts included in lipid-coated lysine products, with alfalfa or corn silage that had 2 different amounts of acidity. Lysine appeared to disassociate from lipid-coated lysine products in a nonlinear manner after mixing with either alfalfa or corn silage at different amounts of acidity. Additionally, silage source and acidity affected amounts of lysine released from lipid-coated lysine products after mixing. In a corresponding experiment, in vitro estimates of lysine available to ruminal microbiota after mixing with alfalfa or corn silage at different amounts of acidity were measured by ammonia release. In vitro measures were conducted with or without monensin to allow estimates of effects of monensin on amounts of lysine released from the 2 lipid-coated lysine products. It is unclear whether in vitro estimates of lysine fermentation from lipid-coated lysine are truly reflective of ruminal degradation of lysine from lipid-coated lysine because amounts of time needed to measure differences between different lysine sources were greater than typical estimates of mean ruminal particulate retention time. Nonetheless, monensin apparently reduced ammonia release from lysine, but ammonia release from lipid-coated lysine did not differ from crystalline lysine. Clearly, methods of manufacture together with physical and chemical characteristics of diet can affect amounts of lysine provided from lipid-coated lysine products to ruminants.

Effect of stocking density on performance, diet selection, total-tract digestion, and nitrogen balance among heifers grazing cool-season annual forages.

Grazing annual cool-season forages after oat grain harvest in South Dakota may allow an opportunity to increase efficient use of tillable land. However, data are limited regarding effects of stocking density on diet selection, nutrient digestion, performance, and N retention by cattle grazing annual cool-season forage. Heifers were blocked by initial BW (261 ± 11.7 kg) and randomly assigned to 1 of 12 paddocks (1.1 ha) to graze a mixture of grass and brassica for 48 d. Each paddock contained 3, 4, or 5 heifers to achieve 4 replicates of each stocking density treatment. Ruminally cannulated heifers were used to measure diet and nutrient intake. Effects of stocking density on diet and nutrient selection were measured after 2, 24, and 46 d of grazing, and BW was measured at the beginning, middle, and end of the experiment as the average of d 1 and 2, d 22 and 23, and d 47 and 48 BW, respectively. Measures of DMI and DM, OM, NDF, and ADF digestion were collected from d 18 to 23. Increased stocking density increased intake of brassica relative to grass on d 24 (quadratic, = 0.02), but increased stocking density decreased (linear, ≤ 0.01) intake of brassica compared with grass on d 48 (stocking density × time, < 0.01). Increased stocking density increased DM (quadratic, < 0.01), OM (quadratic, = 0.01), and NDF (quadratic, = 0.05) digestion, and stocking density tended to increase DMI (quadratic, = 0.07). Additionally, increased stocking density quadratically increased ( = 0.05) N retention but did not affect overall BW gains. Increased stocking density did, however, contribute to linearly decreased ( = 0.05) BW gains from d 1 to 22 of grazing, but BW gains during the latter half of the experiment were greater than BW gains from d 1 to 22. Ruminal concentration of acetate:propionate was least on d 24 of grazing, and ruminal nitrate concentration tended to linearly decrease ( = 0.06) with greater amounts of time on pasture. Ruminal liquid and particulate fill and amounts of VFA were less (quadratic, ≤ 0.01) with greater amounts of time on pasture. Apparently, binary mixtures of brassica and grass planted after oat grain harvest can provide an opportunity to increase efficient use of land by providing forage resources. Increased stocking density may facilitate a more rapid adaptation to and intake of brassica among cattle grazing brassica-grass-based pastures.

Lysine bioavailability among 2 lipid-coated lysine products after exposure to silage.

We conducted 2 experiments to determine lysine bioavailability from 2 lipid-coated lysine products. In an in vitro experiment we mixed each lipid-coated lysine product with either alfalfa- or corn-silage at different amounts of acidity. Scanning electron micrographs indicated that surface structure of each lipid-coated lysine particle was eroded after mixing with silage. Additionally, visual evaluation of scanning electron micrographs suggested that peripheral surface abrasion of lipid-coated lysine may be greater when lipid-coated lysine was mixed with alfalfa silage in comparison to corn silage. In a corresponding experiment, in vivo measures of lysine bioavailability to sheep from 2 lipid-coated lysine products and lysine-HCl were determined after mixing in corn silage. Plasma lysine concentrations increased linearly (P < 0.01) in response to abomasal lysine infusion indicating that our model was sensitive to increases in metabolizable lysine flow. Bioavailability of each lipid-coated lysine source and dietary lysine-HCl were calculated to be 23, 15, and 18%, respectively. Even though each dietary source of lysine increased plasma lysine, rates of increases in plasma lysine from one lipid-coated lysine source (linear; P = 0.20) and lysine-HCl (linear; P = 0.11) were not different from plasma lysine levels supported by diet alone. However, the rate of plasma lysine increase in response to lysine from the other lipid-coated lysine source was greater (P = 0.04) than plasma lysine from feed alone. Nonetheless, the rate of plasma lysine increase in response to lipid-coated lysine did not differ (P ≥ 0.70) from the rate of plasma lysine increase from lysine-HCl. Clearly, methods of manufacture, together with physical and chemical characteristics of diet, can impact amounts of metabolizable lysine provided from lipid-coated lysine products. Direct measures of lysine bioavailability from lipid-coated lysine products after mixing with diets should be based on measurements with the products treated similarly to the method of feeding.

Study protocol for an evaluation of the effectiveness of 'care bundles' as a means of improving hospital care and reducing hospital readmission for patients with chronic obstructive pulmonary disease (COPD).

BACKGROUND: Chronic Obstructive Pulmonary Disease is one of the commonest respiratory diseases in the United Kingdom, accounting for 10% of unplanned hospital admissions each year. Nearly a third of these admitted patients are re-admitted to hospital within 28 days of discharge. Whilst there is a move within the NHS to ensure that people with long-term conditions receive more co-ordinated care, there is little research evidence to support an optimum approach to this in COPD. This study aims to evaluate the effectiveness of introducing standardised packages of care i.e. care bundles, for patients with acute exacerbations of COPD as a means of improving hospital care and reducing re-admissions. METHODS / DESIGN: This mixed-methods evaluation will use a controlled before-and-after design to examine the effect of, and costs associated with, implementing care bundles for patients admitted to hospital with an acute exacerbation of COPD, compared with usual care. It will quantitatively measure a range of patient and organisational outcomes for two groups of hospitals - those who deliver care using COPD care bundles, and those who deliver care without the use of COPD care bundles. These care bundles may be provided for patients with COPD following admission, prior to discharge or at both points in the care pathway. The primary outcome will be re-admission to hospital within 28 days of discharge, although the study will additionally investigate a number of secondary outcomes including length of stay, total bed days, in-hospital mortality, costs of care and patient / carer experience. A series of nested qualitative case studies will explore in detail the context and process of care as well as the impact of COPD bundles on staff, patients and carers. DISCUSSION: The results of the study will provide information about the effectiveness of care bundles as a way of managing in-hospital care for patients with an acute exacerbation of COPD. Given the number of unplanned hospital admissions for this patient group and their rate of subsequent re-admission, it is hoped that this evaluation will make a timely contribution to the evidence on care provision, to the benefit of patients, clinicians, managers and policy-makers. TRIAL REGISTRATION: International Standard Randomised Controlled Trials - ISRCTN13022442 - 11 February 2015.

Critical role of NKT cells in posttransplant alloantibody production.

We previously reported that posttransplant alloantibody production in CD8-deficient hosts is IL-4+ CD4+ T cell-dependent and IgG1 isotype-dominant. The current studies investigated the hypothesis that IL-4-producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8-deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon-γ-dependent and IL-4-independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL-4+ CD4+ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli.

Alloprimed CD8(+) T cells regulate alloantibody and eliminate alloprimed B cells through perforin- and FasL-dependent mechanisms.

While it is well known that CD4(+) T cells and B cells collaborate for antibody production, our group previously reported that CD8(+) T cells down-regulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8(+) T cell-mediated down-regulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8(+) T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to WT transplant recipients. Furthermore, CD8(+) T cells require FasL, perforin and allospecificity to down-regulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8(+) T cells directly induce apoptosis of alloprimed IgG1(+) B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8(+) T cells down-regulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1(+) B cells.

Influence of nitrogen and sulfur intake on bovine uterine pH throughout the luteal phase.

Previous research has reported that diets high in protein and sulfur decreased uterine pH in cattle. The objective of this study was to determine the effect of high N and high S intake on uterine pH. Holstein (n = 15) and Angus-cross (n = 5) heifers (337.5 ± 8.4 kg of BW) were randomly assigned to 1 of 4 diets: control (CON; 13.4% CP and 0.17% S); high nitrogen (HN; CON plus urea supplement); high sulfur (HS; CON plus calcium sulfate); or both high nitrogen and sulfur (HNS). Diets were individually fed at 2.6% of BW on a DM basis using Calan gates and estrus was synchronized to occur on d 13 (d 0 = start of dietary treatment). Blood samples were collected on d -2 and daily (d 1 to 28) at 1400 h to determine concentrations of plasma urea nitrogen (PUN), sulfate (d 1, 4, 8, 12, 16, 20, 24, and 28), and progesterone. Uterine pH was measured on d 16, 20, 24, and 28 (d 3, 7, 11, and 15 of the estrous cycle). There was a treatment, time, and treatment by time interaction (P < 0.01) on concentrations of PUN. There was an effect of treatment (P < 0.01) on concentrations of sulfate, with concentrations being increased in HS compared with CON, HN, and HNS (P < 0.01), and HNS increased compared with CON (P < 0.01) and HN (P < 0.01). Uterine pH was increased in HN and HNS compared with CON (P < 0.02), whereas HS was not different from any treatment (P > 0.11). There was no effect of time (P = 0.26) or treatment by time interaction (P = 0.71) on uterine pH. In summary, uterine pH was increased in HN and HNS compared with CON, whereas HS was intermediate and was associated with increased concentrations of PUN.

Effects of high-sulphur water on hepatic gene expression of steers fed fibre-based diets.

Sulphur-induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is frequently associated with the consumption of high-sulphur (S) water and subsequent poor performance. Currently, there is no economical method for S removal from surface water sources, and alternative water sources are typically neither readily available nor cost-effective. Determination of genes differentially expressed in response to high-S water consumption may provide a better understanding of the physiology corresponding to high dietary S and ultimately lead to the development of treatment and prevention strategies. The objective of this study was to determine changes in gene expression in the liver, an organ important for S metabolism, of fibre-fed steers consuming high-S water. For this study, liver tissues were collected on the final day of a trial from yearling steers randomly assigned to low-S water control (566 mg/kg SO4 ; n = 24), high-S water (3651 mg/kg SO4 ; n = 24) or high-S water plus clinoptilolite supplemented at either 2.5% (n = 24) or 5.0% (n = 24) of diet dry matter (DM). Microarray analyses on randomly selected healthy low-S control (n = 4) and high-S (n = 4; no clinoptilolite) steers using the Affymetrix GeneChip Bovine Genome Array revealed 488 genes upregulated (p < 0.05) and 154 genes downregulated (p < 0.05) in response to the high- vs. low-S water consumption. Real-time RT-PCR confirmed the upregulation (p < 0.10) of seven genes involved in inflammatory response and immune functions. Changes in such genes suggest that ruminant animals administered high-S water may be undergoing an inflammation or immune response, even if signs of sPEM or compromised health are not readily observed. Further study of these, and other affected genes, may deliver new insights into the physiology underlying the response to high dietary S, ultimately leading to the development of treatments for high S-affected ruminant livestock.

Effects of supplemental molybdenum on animal performance, liver copper concentrations, ruminal hydrogen sulfide concentrations, and the appearance of sulfur and molybdenum toxicity in steers receiving fiber-based diets.

Poor performance and S-induced polioencephalomalacia (sPEM) have been observed in ruminant livestock in high-S drinking water regions. No gainful method of removing S from drinking water is available and therefore a feed supplement that negates the effects of high-S water is needed. Our objective was to determine if supplementing Mo improves health and performance of steers administered a high-fiber diet and high-S drinking water. We hypothesized that if the supplemental Mo adequately bound excess S in the rumen, it would not be available at toxic concentrations. Yearling steers (n = 96; 260.0 ± 1.3 kg BW) were stratified by pretrial BW into 12 feedlot pens (n = 8 steers per pen). One of 3 treatments, low-S water (LS; 375 mg SO(4)/L), high-S water (HS; 2,218 mg SO(4)/L), or high-S water plus Mo (HSMO; 2,218 mg SO(4)/L; 187.5 mg Mo/kg DM), were randomly assigned to pens within 4 blocks for a 56-d trial. Body weights were recorded on d -2, -1, 29, 56, and 57, ruminal H(2)S concentrations were measured by rumenocentesis on d -1, 29, and 57, and liver biopsies were performed on d -1 and 57. Performance data were analyzed over the 56-d trial period (overall) as well as over 2 periods: Period 1 (d 0 to d 28) and Period 2 (d 29 to d 56). One case of sPEM was confirmed by the presence of cortical lesions in the HS treatment group. Daily DMI and ADG were affected by treatment and period (P < 0.001) main effects. The LS steers had the greatest (P < 0.05) DMI followed by HS and HSMO steers, respectively. Similar results were observed for ADG. Daily water intake was affected (P < 0.001) by period only, with greater daily water intake in Period 2 than Period 1. Change in hepatic concentrations of Cu, Fe, and Mo over the course of the trial were all affected (P < 0.001) by treatment. Hepatic Cu increased from d 1 to 57 in LS and HS steers but was depleted in HSMO steers. Hepatic Fe and Mo increased in HSMO steers only. Ruminal H(2)S concentrations were affected by treatment (P < 0.021), with greater H(2)S concentrations in HSMO compared with LS and HS steers. Signs of Mo toxicity such as severe diarrhea, loss of body condition, anorexia, changes in hair color, and stiffness in joints were observed in the Mo supplemented steers. These results indicate that added dietary Mo does not adequately bind excess S in the rumen, causing aggravated toxic effects from potentially both the high dietary S and Mo.

Influence of standing estrus before an injection of GnRH during a beef cattle fixed-time AI protocol on LH release, subsequent concentrations of progesterone, and steriodogenic enzyme expression.

Beef cows that exhibit estrus before fixed-time AI have been reported to have increased pregnancy success and increased concentrations of progesterone during the subsequent estrous cycle. Therefore, these experiments were conducted to evaluate if initiation of standing estrus before an injection of GnRH during a fixed-time AI protocol affected LH pulses, subsequent concentrations of progesterone, and luteal steroidogenic enzyme expression. In Experiments 1 and 2, cows were treated with the CO-Synch protocol (100 µg GnRH day -9, 25 mg PGF(2α) day -2, and 100 µg GnRH day 0) and allotted to one of two treatments: 1) cows that initiated estrus before GnRH on day 0 (estrus; n = 5) or 2) cows that did not initiate estrus and were induced to ovulate by the GnRH on day 0 (no estrus; n = 5). In Experiment 1, blood samples were collected at 15-min intervals from 0 to 6 (bleed 1), 12 to 20 (bleed 2), 26 to 34 (bleed 3), and 40 to 48 (bleed 4) h after GnRH. Daily blood samples were collected for 17 d. Initiation of estrus before the GnRH injection had no effect on LH release or the pattern of progesterone increase; however, cows detected in estrus had overall increased (P = 0.002) concentrations of progesterone compared with cows not in estrus. In Experiment 2, estrus was detected with the HeatWatch system. Location and size of the ovulatory follicle was determined on day 0 by transrectal ultrasonography at time of injection with GnRH. Blood samples were collected on days 3, 4, 5, 7, and 9; luteal tissue was collected on day 10 (n = 4 estrus and n = 9 no estrus) from corpus luteum (CL) originating from similar-sized follicles (13.0 to 16.0 mm). Total cellular RNA was extracted, and relative mRNA levels were determined by real-time reverse transcription PCR and corrected for GAPDH. There was no effect of estrus on CL weight or concentrations of progesterone. In addition, there was no effect of estrus, follicle size, or CL weight on luteal expression of LH receptor, StAR, CYP11A1, or 3ßHSD. However, there was a correlation between follicle size and CL weight (P = 0.01; R(2) = 0.43); for every increase of 1 mm in follicle size, CL weight increased by 1.5 g. In summary, estrus did not influence release of LH, CL weight, progesterone concentrations, or expression of steriodogenic enzymes. However, as follicle size increased, CL weight increased; therefore, both follicle size and CL weight were associated with progesterone concentrations.

Influence of feeding various quantities of wet and dry distillers grains to finishing steers on carcass characteristics, meat quality, retail-case life of ground beef, and fatty acid profile of longissimus muscle.

Two hundred forty Angus crossbred steers were used to determine the influence of feeding various quantities of wet and dry distillers grains to finishing steers on carcass characteristics, meat quality, retail-case life of ground beef, and fatty acid profile of LM. Three replications of 5 dietary treatments were randomly applied to 15 pens in each of 2 yr. A finishing diet containing dry-rolled corn, soybean meal, and alfalfa hay was fed as the control diet. Wet distillers grains with solubles (DGS) or dry DGS was added to the finishing diets at either 20.0 or 40.0% of the dietary DM to replace all soybean meal and part of the cracked corn in treatment diets. Carcasses of steers fed DGS had greater (P < 0.05) fat thickness (1.47 vs. 1.28 cm), greater (P < 0.05) USDA yield grades (3.23 vs. 2.94), and smaller (P < 0.05) percentage of yield grades 1 and 2 (41.1 vs. 60.4%) than carcasses of steers fed the control diet. Longissimus muscle from steers fed dry DGS had greater (P < 0.05) ultimate pH values (5.52 vs. 5.49) than LM from steers fed wet DGS. Ground beef from steers fed DGS had greater (P < 0.05) concentrations of α-tocopherol (1.77 vs. 1.43 µg/g) than ground beef from steers fed the control diet. Ground beef from steers fed 40% DGS had greater (P < 0.05) thiobarbituric acid-reactive substances (2.84 vs. 2.13 mg/kg) on d 2 of retail display than ground beef from steers fed 20% DGS. Longissimus muscle of steers fed DGS had less (P < 0.05) C17:0 and more (P < 0.05) C18:0, C18:1t, C16:1c9, C18:2c9c12 (where t is trans and c is cis), and total PUFA than LM of steers fed the control diet. Feedlot steers fed DGS may need to be marketed earlier than normal to avoid excess external fat and carcasses with a greater numerical yield grade. These data suggest feeding DGS to finishing steers will have no adverse or beneficial effects on glycolytic variables (dark cutters), retail display life of ground beef, or meat tenderness. However, beef from cattle finished on diets containing DGS will likely have a greater proportion of PUFA and therefore may be more susceptible to oxidative rancidity.

Effects of high-sulfur water and clinoptilolite on health and growth performance of steers fed forage-based diets.

Sulfur-induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is associated with consumption of diets with increased S (high-S). High-S water is commonly found in many western states and is a major source of dietary S for grazing cattle. Consumption of high-S water has been associated with sPEM and decreased performance. Identification of a feed supplement that would counteract the negative effects of high-S water would decrease the incidence of sPEM and prevent performance reductions in regions with problematic water sources. The objectives of this study were to 1) determine the effects of administering high-S drinking water to forage-fed feedlot steers on health and performance, and 2) determine the effectiveness of clinoptilolite, a clay mineral with increased cation-exchange capacity, in negating the effects of high-S drinking water. Yearling steers (n = 96; 318.2 +/- 2.1 kg of BW) were randomly assigned to 1 of 4 treatments for a 77-d trial period: control with low-S water (566 mg of SO(4)/L), high-S water (3,651 mg of SO(4)/L), or high-S water plus clinoptilolite supplemented at 2.5 or 5.0% of the diet DM. Feed and water consumption were measured daily, and all steers were weighed on d -2, -1, 29, 53, 76, and 77. Plasma samples were collected on d 0, 58, and 77, and liver samples on d 0 and 77. There was a greater (P or= 0.546) in ADG or G:F were observed. Plasma Cu decreased (P = 0.029) to a greater magnitude in high-S water steers than the control steers over the 77-d trial period. Mineral analyses of hepatic tissue from randomly selected healthy steers from each treatment group (n = 10 per treatment) showed an interaction (P

Dose comparison of single versus double dose in contrast-enhanced magnetic resonance angiography of the renal arteries: intra-individual cross-over blinded trial using Gd-DTPA.

This study was planned as an open-label, single-centre trial with blinded evaluations by two independent radiologists, aimed at the intra-individual comparison of single-dose and double-dose Gd-DTPA-enhanced MRA in the renal arterial territory. Ten healthy volunteers were included in the study. Renal MRAs were carried out on a clinical 1.5-T MR system using a body phased-array coil. Images were acquired with three-dimensional fast spoiled gradient echo sequence. Contrast agent was injected with a power injector keeping the injection time constant for single dose and double dose. Both readers found at least 96% of vascular segments evaluable. Median overall image quality was rated excellent, and diagnostic confidence was rated confident. No statistically significant difference between the dosage groups could be demonstrated. Signal intensity, SNR and CNR were significantly higher for the double-dose group. Our results demonstrate that while a double dose of contrast agent increases SNR, it does not lead to further improvement in visual and perceptual image quality. A single dosage of approximately 0.1 mmol/kg bw Gd-DTPA may be the preferable dosage to demonstrate the renal arteries.

Mechanisms mediating oestradiol modulation of the developing brain.

The brain has been known to be a sensitive target organ for the permanent organisational effects of gonadal steroids for close to 50 years. Recent advances have revealed a variety of unexpected cellular mechanisms by which steroids impact on the synaptic profile of hypothalamic nuclei critical to the control of reproduction. This review focuses on three in particular: 1) prostaglandins in the masculinisation of the preoptic area and control of male sexual behaviour; 2) GABA in the arcuate nucleus and potential control of the anterior pituitary; and 3) non-genomic activation of phosphotydolinositol 3 (PI3) kinase and glutamate in the ventromedial nucleus, which is relevant to the control of female reproductive behaviour. The importance of cell-to-cell communication, be it between neurones or between neurones and astrocytes, is highlighted as an essential principle for expanding the impact of steroids beyond those cells that express nuclear receptors.

Uptake of zinc from zinc sulfate and zinc proteinate by ovine ruminal and omasal epithelia.

Uptake and transport of Zn from (65)Zn-labeled ZnSO(4) and Zn proteinate (ZnProt) by ruminal and omasal epithelia were examined by using a parabiotic chamber system. Uptake was measured during a 4-h incubation with 10, 20, or 200 microM Zn as ZnSO(4) or ZnProt in the mucosal buffer (pH 6.0, Krebs-Ringer phosphate). Zinc uptake and transport were also evaluated after simulated ruminal digestion. Buffered ruminal fluid contained a feed substrate and 10 or 200 microM added Zn as ZnSO(4) or ZnProt. In a preliminary experiment, uptake of Zn by omasal tissue was low; thus, the remaining experiments were conducted solely with ruminal epithelium. Incubations to determine the effect of time on Zn uptake from mucosal buffer containing 20 microM added Zn as ZnSO(4) or ZnProt resulted in increased (P < 0.01) Zn uptake as incubation time increased from 30 to 240 min. Zinc uptake was also greater (P = 0.02) from mucosal buffer containing ZnProt compared with ZnSO(4). Zinc uptake from incubations containing 10 or 200 microM was affected by source x concentration (P = 0.05) and concentration x time (P < 0.01) interactions. With 10 microM Zn, uptake was not influenced by Zn source, whereas when 200 microM Zn was added, Zn uptake from ZnProt was greater than from ZnSO(4). Increasing incubation time resulted in increased Zn uptake with 200 microM Zn in the mucosal buffer; however, with 10 microM Zn, uptake did not change after 30 min. After simulated ruminal fermentation, the proportion of Zn in a soluble form was influenced by a source x concentration interaction (P = 0.03). After 18 h of incubation, the proportion of Zn that was soluble was not different between ZnProt and ZnSO(4) in buffered ruminal fluid that contained 10 microM added Zn, but was greater for ZnProt compared with ZnSO(4) with 200 microM Zn in the incubation. Zinc uptake from the aqueous fractions of simulated ruminal digestions containing 200 microM added Zn was greater (P < 0.01) than from those containing 10 microM added Zn. Zinc transport, based on detection of (65)Zn in serosal buffer, did not occur in any of the experiments. The results of the current experiments suggest that absorption of Zn into the bloodstream does not occur from the ruminant foresto-mach; however, Zn uptake occurs in ruminal tissue and is greater from ZnProt than from ZnSO(4).

Exploration of prostanoid receptor subtype regulating estradiol and prostaglandin E2 induction of spinophilin in developing preoptic area neurons.

The prostaglandin E2 (PGE2) mediates estradiol-induced masculinization of sexual behavior in the rat during a perinatal sensitive period. PGE2 induces formation of dendritic spines on preoptic area (POA) neurons and this synaptic pattern change is associated with the ability to express male sexual behavior as an adult. Whether PGE2 is released from astrocytes or neurons in the developing POA is unknown. To further understanding of how PGE2 induces dendritic spine formation at the cellular level, we have explored the PGE2 receptor subtype mediating this response. There are four receptors for PGE2, EP1, EP2, EP3 and EP4, each having unique but interacting signal transduction profiles. Treatment of newborn female rats with the EP receptor agonists iloprost, butaprost and sulprostone indicated that stimulation of both the EP2 and EP3 receptors significantly increased spinophilin, a protein whose levels positively correlate to the presence of dendritic spines and masculinization of the POA. Use of antisense oligonucleotides against the mRNA for each receptor reveals that either EP2 or EP3 receptor knockdown reduces spinophilin in PGE2- or estradiol-treated females, whereas reducing EP1 or EP4 receptor levels by the same means has a smaller but also significant effect. A developmental profile of EP receptor expression indicates EP1 in particular is elevated for the first few days of life, corresponding to the critical period for masculinization, whereas mRNA levels for the other three receptors remain relatively constant.

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor.

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (alpha v beta6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an alpha v beta6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-zeta. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin alpha v beta6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.