Ferumoxytol-enhanced MRI assessment of venous Thrombus resolution and macrophage content in a murine deep vein thrombosis model.

BACKGROUND: Imaging evaluation of acute deep vein thrombosis (DVT) or post-thrombotic syndrome (PTS) in animal or clinical models is limited to anatomical assessment of the location and extent of thrombi. We hypothesize that Fe-MRI, used to evaluate macrophage content in other inflammatory diseases, can be useful to evaluate the thromboinflammatory features after DVT over time. METHODS: Nineteen wild-type CD-1 mice underwent surgical IVC ligation to induce DVT. Mice received either saline or 5 mg/kg of 14E11, a Factor XI inhibitor, before the procedure. Fe-MRI was performed on days 6-7 after ligation to evaluate thrombus volume, perfusion, and macrophage content via T2-weighted images. Mice were euthanized at days 3-15 after surgery. The thrombi and adjacent vein walls were excised, weighed, formalin-fixed, and paraffin-embedded for immunohistological analysis. Specimens were stained with specific antibodies to evaluate macrophage content, collagen deposition, neovascularization, and recanalization. Significance was determined using the Mann-Whitney U or Student's t-test. RESULTS: After IVC-ligation in control mice, thrombus weights decreased by 59 % from day 3 to 15. Thrombus volumes peaked on day 5 before decreasing by 85 % by day 13. FXI inhibition led to reduced macrophage content in both thrombi (p = .008) and vein walls (p = .01), decreased thrombus volume (p = .03), and decreased thrombus mass (p = .01) compared to control mice. CCR2+ staining corroborated these findings, showing significantly reduced macrophage presence in the thrombi (p = .002) and vein wall (p = .002). CONCLUSIONS: Fe-MRI T2 relaxation times can be used to characterize and quantify post-thrombotic changes of perfusion, macrophage content, and thrombus volume over time in a surgical mouse model of venous thrombosis. This approach could lead to better quantification of in vivo inflammation correlating monocyte and macrophage content within resolving thrombi and veins and may serve as a useful tool for research and clinically in the evaluation of the post-thrombotic environment.

Thrombogenicity of biodegradable metals.

Biodegradable metals offer a promising means to ameliorate many of the long-term risks associated with vascular devices made of conventional biostable stent metals. While numerous biodegradable metal alloys have been developed and characterized in animal models, knowledge of their blood reactivity and thrombogenicity remains unknown. Metal hemocompatibility is particularly valuable because current generation drug-eluting stents pose a significant long-term thrombosis risk. In this study, four pure metals, widely used as degradable base materials (Fe, Zn, Mg, and Mo), and three alloys commonly used in cardiovascular devices [NiTi, CoCr, and stainless steel (SS)] were evaluated. This work examined how each of these metals activate platelets, coagulation factors, and inflammation using in vitro hemocompatibility assays and a clinically relevant ex vivo non-human primate arteriovenous shunt model. Testing found that while all metals promoted a downstream activation of platelets and coagulation in flowing whole blood, platelet and fibrin attachment to Mg was markedly reduced. Additionally, Fe and Mo trended toward higher platelet attachment and contact pathway activation. Overall, the results suggest that Mg may delay clot initiation, but not eliminate clot formation, indicating the importance of understanding thrombosis in Mg alloys that are currently being developed for clinical use as biodegradable stents.

Development of Coagulation Factor XII Antibodies for Inhibiting Vascular Device-Related Thrombosis.

INTRODUCTION: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. OBJECTIVE: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. METHODS: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. RESULTS: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. CONCLUSION: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

Guidance for elective single-embryo transfer should be applied to frozen embryo transfer cycles.

PURPOSE: To provide clinicians with data showing the benefits of transferring a single blastocyst in frozen embryo transfer (FET) cycles so that they may counsel their patients accordingly. METHODS: This is a closed cohort study of 678 FET cycles occurring between January 2011 and December 2017 in a private IVF laboratory and associated physicians' practice. Patients included in the analysis were less than 38 years of age at oocyte collection, had at least two vitrified blastocysts, and were undergoing their first autologous FET cycle. The patients were categorized into four groups after they had chosen either elective single-embryo transfer (eSET) or double-embryo transfer (eDET). Outcomes for eSET and eDET were compared within groups of patients having freeze-all IVF cycles (PGT-A patient vs. non-PGT-A patient) and fresh IVF transfer groups (negative outcome vs. pregnant/delivered in fresh cycle). Main outcome measures of the study were live birth, multiple pregnancy, and implantation rates. RESULTS: There were no statistically significant differences observed in live birth rates for eSET (54-62%) vs. eDET (54-66%) (P = 0.696-1.000) in the four patient groups evaluated. Multiple pregnancy rates were significantly decreased in all eSET groups (0-3%), compared with eDET groups (24-65%) (P = 0.0001-0.037). CONCLUSIONS: This data shows that transfer of a single vitrified-warmed blastocyst maintains live birth rates, while decreasing multiple pregnancies, and may become more acceptable to physicians and patients.

Predicting the digestible energy of corn determined with growing swine from nutrient composition and cross-species measurements.

The DE values of corn grain for pigs will differ among corn sources. More accurate prediction of DE may improve diet formulation and reduce diet cost. Corn grain sources ( = 83) were assayed with growing swine (20 kg) in DE experiments with total collection of feces, with 3-wk-old broiler chick in nitrogen-corrected apparent ME (AME) trials and with cecectomized adult roosters in nitrogen-corrected true ME (TME) studies. Additional AME data for the corn grain source set was generated based on an existing near-infrared transmittance prediction model (near-infrared transmittance-predicted AME [NIT-AME]). Corn source nutrient composition was determined by wet chemistry methods. These data were then used to 1) test the accuracy of predicting swine DE of individual corn sources based on available literature equations and nutrient composition and 2) develop models for predicting DE of sources from nutrient composition and the cross-species information gathered above (AME, NIT-AME, and TME). The overall measured DE, AME, NIT-AME, and TME values were 4,105 ± 11, 4,006 ± 10, 4,004 ± 10, and 4,086 ± 12 kcal/kg DM, respectively. Prediction models were developed using 80% of the corn grain sources; the remaining 20% was reserved for validation of the developed prediction equation. Literature equations based on nutrient composition proved imprecise for predicting corn DE; the root mean square error of prediction ranged from 105 to 331 kcal/kg, an equivalent of 2.6 to 8.8% error. Yet among the corn composition traits, 4-variable models developed in the current study provided adequate prediction of DE (model ranging from 0.76 to 0.79 and root mean square error [RMSE] of 50 kcal/kg). When prediction equations were tested using the validation set, these models had a 1 to 1.2% error of prediction. Simple linear equations from AME, NIT-AME, or TME provided an accurate prediction of DE for individual sources ( ranged from 0.65 to 0.73 and RMSE ranged from 50 to 61 kcal/kg). Percentage error of prediction based on the validation data set was greater (1.4%) for the TME model than for the NIT-AME or AME models (1 and 1.2%, respectively), indicating that swine DE values could be accurately predicted by using AME or NIT-AME. In conclusion, regression equations developed from broiler measurements or from analyzed nutrient composition proved adequate to reliably predict the DE of commercially available corn hybrids for growing pigs.

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax.

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

Structural biology of the Bcl-2 family and its mimicry by viral proteins.

Intrinsic apoptosis in mammals is regulated by protein-protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.

The restricted binding repertoire of Bcl-B leaves Bim as the universal BH3-only prosurvival Bcl-2 protein antagonist.

B-cell lymphoma-2 (Bcl-2) proteins mediate intrinsic-, or mitochondrial-, initiated apoptosis. We have investigated the structure and function of the least characterized Bcl-2 family member, Bcl-B, solving the crystal structure of a Bcl-B:Bim complex to 1.9 Å resolution. Bcl-B is distinguished from other Bcl-2 family members through an insertion of an unstructured loop between helices α5 and α6. Probing Bcl-B interactions with Bcl-2 homology (BH)3 motifs using a combination of biophysical- and cell-based assays revealed a unique BH3-only protein binding profile. Bcl-B has high-affinity interactions with Bim and Bik only. Our results not only delineate the mode of action of Bcl-B but also complete our understanding of the specific interactions between BH3-only proteins and their prosurvival Bcl-2 counterparts. Notably, we conclude that Bim is the universal prosurvival antagonist as no other BH3-only protein binds all six prosurvival proteins and that Mcl-1 and Bcl-x(L) form a distinct prosurvival dyad.

Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies.

Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8(+) conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.

Comparison of broiler performance and carcass yields when fed transgenic maize grain containing event DP-O9814O-6 and processed fractions from transgenic soybeans containing event DP-356O43-5.

The performance of broilers fed diets containing maize grain from event DP-Ø9814Ø-6 (98140; gat4621 and zm-hra genes) and processed fractions (meal, hulls, and oil) from soybeans containing event DP-356Ø43-5 (356043; gat4601 and gm-hra genes) was evaluated in a 42-d feeding study. Diets were produced with nontransgenic maize grain and soybean fractions from controls with comparable genetic backgrounds to 98140 and 356043 (control), 98140 maize and 356043 soybean (98140 + 356043), or 3 commercially available nontransgenic maize and soybean combinations. Ross 708 broilers (n = 120/group; 50% male, 50% female) were fed diets in 3 phases: starter (d 0 to 21), grower (d 22 to 35), and finisher (d 36 to 42). Starter diets contained (on average) 63% maize and 28% soybean meal, grower diets 66% maize and 26% soybean meal, and finisher diets 72% maize and 21% soybean meal; soybean hulls and oils were held constant at 1.0 and 0.5%, respectively, across all diets in all phases. Weight gain, feed intake, and mortality-adjusted feed efficiency were calculated for d 0 to 42. Standard organ and carcass yield data were collected on d 42. Data were analyzed using a mixed model ANOVA with differences between control and 98140 + 356043 group means considered significant at P < 0.05. Reference group data were used only to estimate experimental variability and to generate tolerance intervals. No significant differences were observed in weight gain, mortality, mortality-adjusted feed efficiency, organ yields, or carcass yields between broilers consuming diets produced with 98140 + 356043 and those consuming diets produced with control maize and soybean fractions. All values of response variables evaluated in the control and 98140 + 356043 groups fell within calculated tolerance intervals. Based on these results, it was concluded that the combination of genetically modified 98140 maize and 356043 soybean fractions was nutritionally equivalent to nontransgenic maize and soybean controls with comparable genetic backgrounds.

Performance of lactating dairy cows fed corn as whole plant silage and grain produced from genetically modified corn containing event DAS-59122-7 compared to a nontransgenic, near-isogenic control.

The nutritional equivalency of grain plus whole plant silage from genetically modified corn plants containing the DAS-59122-7 (59122) event expressing the Cry34Ab1 and Cry35Ab1 proteins to grain and silage from a near-isogenic corn hybrid without this trait (control) was assessed using lactating dairy cows. Corn plants with event 59122 are resistant to western corn rootworm and tolerant to the herbicide active ingredient glufosinate-ammonium. Effects on feed intake, milk production, and milk composition were determined. The 59122 grain and the control grain were produced in 2005 from isolated plots in Richland, Iowa. Whole plant corn silage for the 59122 and control treatments were grown in isolated plots at the Kansas State University Dairy Center and ensiled in Ag-Bags. Thirty lactating Holstein cows blocked by lactation number, day of lactation, and previous energy-corrected milk production were used in a switchback design. All cows were fed diets that contained 22.7% grain plus 21.3% whole plant silage from either the 59122 or the control hybrid, in addition to 21% wet corn gluten feed, 12.3% protein mix, 8.0% whole cottonseed, and 14.7% alfalfa hay. Each period of the switchback trial included 2 wk for diet adjustment followed by 4 wk for data and sample collection. Milk samples (a.m. and p.m.) collected from 2 consecutive milkings of each collection wk were analyzed for fat, protein, lactose, solids-not-fat, milk urea nitrogen, and somatic cell count. Percentages of milk fat, protein, lactose, and solids-not-fat were not affected by dietary treatment. Yields of milk, 4% fat-corrected milk, energy-corrected milk, solids-corrected milk, and the concentrations and yields of milk fat, milk protein, milk solids, and milk lactose were not significantly different between treatments. Efficiencies of milk, fat-corrected milk, energy-corrected milk, and solids-corrected milk production also were not different when cows were fed crops from 59122 than when they were fed the control hybrid. Milk production efficiency averaged 1.48 and 1.50 kg/kg of dry matter intake for cows fed diets containing the control and 59122 corn, respectively. These data indicate that the nutritional value for milk production was not different between a diet containing grain plus whole plant corn silage produced from a 59122 corn hybrid versus a diet containing grain and corn silage from its near-isogenic control corn hybrid.

Nutritional equivalency evaluation of transgenic maize grain from event DP-O9814O-6 and transgenic soybeans containing event DP-356O43-5: laying hen performance and egg quality measures.

The objective of this study was to compare the nutritional performance of laying hens fed maize grain from event DP-Ø9814Ø-6 (98140; gat4621 and zm-hra genes) and processed soybean meal from soybeans containing event DP-356Ø43-5 (356043; gat4601 and gm-hra genes), individually or in combination, with the performance of hens fed diets containing nontransgenic maize and soybean meal. Healthy pullets (n = 216) placed in cages (3 hens/cage) were randomly assigned to 9 dietary treatments (8 cages/treatment): nontransgenic controls 1, 2, and 3 (comparable genetic background controls for 98140, 356043, and 98140 + 356043, respectively); reference 1, reference 2, and reference 3 (commercially available nontransgenic maize-soybean meal sources); and 98140 (test 1), 356043 (test 2), and 98140 + 356043 (test 3). The experiment was divided into three 4-wk phases (24 to 28 wk, 28 to 32 wk, and 32 to 36 wk of age), during which time hens were fed mash diets. Performance (BW, feed intake, and egg production) and egg quality data were collected. Data were analyzed using a mixed model ANOVA; differences between the control and respective test group means were considered significant at P < 0.05. Data generated from the reference groups were used only in the estimation of experimental variability and in generating the tolerance interval. Body weight and BW gain, egg production, and production efficiency for hens fed the test diets were similar to the respective values for hens fed the corresponding control diets. Haugh unit measures and egg component weights were similar between the respective test and control groups, and no differences were observed in quality grades or crack measures. All observed values of the control and test groups were within the calculated tolerance intervals. This research indicates that the performance and egg quality of hens fed diets containing 98140 maize grain, 356043 soybean meal, or a combination of the 2 was comparable with that of hens fed diets formulated with nontransgenic maize grain or soybean meal control diets with comparable genetic backgrounds.

Evaluation of corn grain with the genetically modified input trait DAS-59122-7 fed to growing-finishing pigs.

A growth performance experiment was conducted to assess the feeding value of a double-stacked transgenic corn grain for growing-finishing pigs. The genetically modified corn grain contained event DAS-59122-7, which expresses the Cry34/35Ab1 binary insecticidal protein for the control of corn rootworm. This modified transgenic grain is resistant to western corn rootworm and is also tolerant to herbicides containing the active ingredient glufosinate-ammonium. The modified grain (59122), a nontransgenic near-isoline grain (control corn), and a commercial corn (Pioneer brand hybrid 35P12) were grown in a 2005 production trial in individually isolated plots that were located 201 m apart. A total of 108 pigs were allotted to corn-soybean meal diets containing 1 of the 3 grains as the sole source of corn. There were 3 pigs per pen and 12 replicate pens per treatment. Pigs were fed grower diets from 37 to 60 kg, early finisher diets from 60 to 90 kg, and late finisher diets from 90 to 127 kg. Within each phase, data for ADG, ADFI, and G:F were calculated. At the conclusion of the experiment, pigs were slaughtered and data for carcass quality were collected. Differences between 59122 and the control corn were evaluated, with statistical significance at P<0.05. No differences in ADG, ADFI, or G:F between pigs fed the control corn and pigs fed the modified corn were observed during the grower, early finisher, or late finisher phases. For the entire experimental period, no difference between pigs fed the control and the 59122 corn were observed for final BW (128.9 vs. 127.1 kg), ADG (1.02 vs. 1.00 kg), ADFI (2.88 vs. 2.80 kg), or G:F (0.356 vs. 0.345 kg/kg). Likewise, no differences in dressing percentage (76.48 vs. 76.30%), LM area (49.8 vs. 50.4 cm(2)), 10th-rib back fat (2.20 vs. 2.12 cm), and carcass lean content (52.9 vs. 53.4%) were observed between pigs fed the control and the 59122 corn grain. It was concluded that the nutritional value of the modified transgenic corn grain containing event DAS-59122-7 was similar to that of the nontransgenic near-isoline control.

Comparison of broiler performance when fed diets containing event DP-3O5423-1, nontransgenic near-isoline control, or commercial reference soybean meal, hulls, and oil.

DP-3Ø5423-1 (305423) is a genetically modified soybean that was produced by biolistic insertion of the gm-fad2-1 gene fragment and gm-hra genes into the germline of soybean seeds. Expression of gm-fad2-1 results in greater concentrations of oleic acid (18:1) by suppressing expression of the endogenous FAD2-1 gene, which encodes an n-6 fatty acid desaturase enzyme that catalyzes desaturation of 18:1 to linoleic acid (18:2). The GM-HRA protein expressed by the gm-hra gene is a modified version of the soybean acetolactate synthase enzyme that is used as a selectable marker during transformation. A 42-d feeding trial was conducted with broiler chickens to compare the nutritional performance of 305423 soybeans with nontransgenic soybeans. Diets were prepared using processed fractions (meal, hulls, and oil) from 305423 soybean plants. For comparison, additional diets were produced with soybean fractions obtained from a nontransgenic near-isoline (control) and nontransgenic commercial Pioneer brand varieties (93B86, 93B15, and 93M40). Diets were fed to Ross x Cobb broilers (n = 120/group, 50% male and 50% female) in 3 phases. Starter, grower, and finisher diets contained 26.5, 23, and 21.5% soybean meal, respectively. Soybean hulls and oil were added at 1.0 and 0.5%, respectively, across all diets in each phase. No statistically significant differences were observed in growth performance (BW, mortality, feed efficiency), organ yield (liver and kidney), or carcass yield (breast, thigh, leg, wing, and abdominal fat) variables between broilers consuming diets prepared with isolated fractions from 305423 or near-isoline control soybean. Additionally, all performance and carcass variables from control and 305423 soybean treatment groups fell within tolerance intervals constructed for each response variable using data from broilers fed diets prepared with reference soybean fractions. Based on the results from this study, it was concluded that 305423 soybeans were nutritionally equivalent to non-transgenic control soybeans with a comparable genetic background.

Comparison of broiler performance and carcass yields when fed diets containing transgenic maize grains from event DP-O9814O-6 (Optimum GAT), near-isogenic control maize grain, or commercial reference maize grains.

A genetically modified maize (Zea mays L.) line that contains the Optimum GAT trait (event DP-Ø9814Ø-6; 98140) was produced by integration of the gat4621 and zm-hra genes. The expressed GAT4621 and ZM-HRA proteins confer tolerance to the herbicidal active ingredient glyphosate and acetolactate synthase-inhibiting herbicides, respectively. The objective of this study was to compare the nutritional performance of 98140 maize grain to nontransgenic maize grain in a 42-d feeding trial in broiler chickens. Diets were prepared using grain from untreated 98140 plants and from plants treated with an in-field application of herbicides (98140 + Spray). For comparison, additional diets were produced with maize grain obtained from the nontransgenic near-isogenic control (control) and nontransgenic commercial reference Pioneer brand hybrids 33J56, 33P66, and 33R77. Diets were fed to Ross x Cobb broilers (n = 120/group, 50% male and 50% female) in 3 phases: starter, grower, and finisher containing 58.5, 64, and 71.5% maize grain, respectively. No statistically significant differences were observed in mortality, growth performance variables, or carcass and organ yields between broilers consuming diets produced with maize grains from unsprayed or sprayed 98140 and those consuming diets produced with near-isogenic control maize grain. Additionally, all performance and carcass variables from control, 98140, and 98140 + Spray test maize treatment groups were within tolerance intervals constructed using data from reference maize groups. Based on these results, it was concluded that 98140 maize grain (unsprayed or sprayed with a herbicide mixture) was nutritionally equivalent to nontransgenic control maize with comparable genetic background.

Performance of laying hens fed diets containing DAS-59122-7 maize grain compared with diets containing nontransgenic maize grain.

An experiment using 216 Hy-Line W-36 pullets was conducted to evaluate transgenic maize grain containing the cry34Ab1 and cry35Ab1 genes from a Bacillus thuringiensis (Bt) strain and the phosphinothricin ace-tyltransferase (pat) gene from Streptomyces viridochromogenes. Expression of the cry34Ab1 and cry35Ab1 genes confers resistance to corn rootworms, and the pat gene confers tolerance to herbicides containing glufosinate-ammonium. Pullets (20 wk of age) were placed in cage lots (3 hens/cage, 2 cages/lot) and were randomly assigned to 1 of 3 corn-soybean meal dietary treatments (12 lots/treatment) formulated with the following maize grains: near-isogenic control (control), conventional maize, and transgenic test corn line 59122 containing event DAS-59122-7. Differences between 59122 and control group means were evaluated with statistical significance at P < 0.05. Body weight and gain, egg production, egg mass, and feed efficiency for hens fed the 59122 corn were not significantly different from the respective values for hens fed diets formulated with control maize grain. Egg component weights, Haugh unit measures, and egg weight class distribution were similar regardless of the corn source. This research indicates that performance of hens fed diets containing 59122 maize grain, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with near-isogenic corn grain.

Performance of lactating dairy cows fed silage and grain from a maize hybrid with the cry1F trait versus its nonbiotech counterpart.

Effects of feeding grain and maize silage from a non-Bt maize and a variety of Bt maize that contains cry1F (event TC1507, event DAS-Ø15Ø7-1), a gene that provides maize with insect resistance, on the health and performance of dairy cows were evaluated. In a crossover trial, 20 lactating Holstein cows were assigned to each of 2 dietary treatment groups and fed diets containing whole-plant maize silage plus maize grain from TC1507 or its near-isoline counterpart (control). Each period of the crossover trial lasted 28 d and was preceded by a 7-d adjustment period. To minimize variability due to stage of lactation, 2 blocks of 10 cows at 90 to 130 d of lactation at the start of the trial were used. Within each dietary treatment, 10 cows were from each of 2 genetic selection lines (high and average fat plus protein predicted transmitting ability). Diets were formulated to be isocaloric and isonitrogenous. Dry matter intake and daily production of milk, fat, protein, lactose, nonfat solids, and total solids did not differ between cows fed the TC1507 diet and cows fed the control diet. Furthermore, milk from cows in different dietary treatment groups did not differ in milk urea nitrogen concentration or somatic cell count. For milk fat percentage, a significant dietary treatment by genetic group interaction was detected although overall yield of milk and solids-corrected milk did not differ with diet. Physical measures of cow health including body weight, body condition score, temperature, pulse, and respiration rate were collected weekly; dietary treatment group means for these measures were not different. Blood chemistry and hematological analyses were conducted using blood samples collected from cows before the start of the trial and at the end of each period. Overall, the TC1507 and control groups did not differ in any of these indices of health status. Further, hematological profiles for cows in the dietary treatment groups were not different. In summary, no differences were detected in milk production, milk composition, or cow health as indicated by physical measures, blood chemistry, and hematological analyses between dairy cows fed diets containing maize grain plus whole-plant maize silage from TC1507 and dairy cows fed grain plus silage from its near-isoline counterpart.

Comparison of broiler performance when fed diets containing event DP-356Ø43-5 (Optimum GAT), nontransgenic near-isoline control, or commercial reference soybean meal, hulls, and oil.

Event DP-356Ø43-5 (356043; Optimum GAT) is a genetically modified soybean (Glycine max) that was produced by insertion of the gat4601 and gm-hra genes. The expression products of these genes are the glyphosate acetyltransferase 4601 and acetolactate synthase proteins, respectively. Expression of the glyphosate acetyltransferase 4601 protein confers tolerance in planta to the herbicidal active ingredient glyphosate, whereas expression of the acetolactate synthase protein confers tolerance to sulfonylurea and imidazolinone herbicides. The objective of this study was to compare the nutritional equivalence of 356043 soybeans to nontransgenic soybeans in a 42-d feeding trial in broiler chickens. Diets were prepared using processed fractions (meal, hulls, and oil) from untreated 356043 soybean plants or from soybean plants treated with a mixture of glyphosate, chlorimuron, and thifensulfuron (356043 + Gly/SU). For comparison, additional diets were produced with soybean fractions obtained from a nontransgenic near-isoline (control; 091) and nontransgenic commercial Pioneer varieties (93B86, 93B15, and 93M40). Diets were fed to Ross x Cobb broilers (n = 120/group, 50% male and 50% female) in 3 phases. Starter diets contained 30% soybean meal, grower diets 26% soybean meal, and finisher diets 21.5% soybean meal. Soybean hulls and oil were added at 1.0 and 0.5%, respectively, across all diets in each phase. No statistically significant differences were observed in mortality, growth performance variables, or carcass and organ yields between broilers consuming diets produced with 356043 or 356043 + Gly/SU soybean fractions and those consuming diets produced with near-isoline control soybean fractions. Additionally, all performance and carcass variables from control, 356043, and 356043 + Gly/SU soybean treatment groups fell within the tolerance intervals constructed using data from reference soybean groups. Based on the results from this study, it was concluded that 356043 soybean was nutritionally equivalent to nontransgenic control soybean with a comparable genetic background.

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets.

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance.

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.