Multi-modal imaging for assessment of tissue-engineered bone in a critical-sized calvarial defect mouse model.

Tissue-engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end-point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non-invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical-sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near-infrared (NIR) optical imaging with IRDye® 800CW BoneTagTM (800CW BT, a bone-specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi-modal non-invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd.

Swine manure injection with low-disturbance applicator and cover crops reduce phosphorus losses.

Injection of liquid swine manure disturbs surface soil so that runoff from treated lands can transport sediment and nutrients to surface waters. We determined the effect of two manure application methods on P fate in a corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] production system, with and without a winter rye (Secale cereale L.)-oat (Avena sativa L.) cover crop. Treatments included: (i) no manure; (ii) knife injection; and (iii) low-disturbance injection, each with and without the cover crop. Simulated rainfall runoff was analyzed for dissolved reactive P (DRP) and total P (TP). Rainfall was applied 8 d after manure application (early November) and again in May after emergence of the corn crop. Manure application increased soil bioavailable P in the 20- to 30-cm layer following knife injection and in the 5- to 20-cm layer following low-disturbance injection. The low-disturbance system caused less damage to the cover crop, so that P uptake was more than threefold greater. Losses of DRP were greater in both fall and spring following low-disturbance injection; however, application method had no effect on TP loads in runoff in either season. The cover crop reduced fall TP losses from plots with manure applied by either method. In spring, DRP losses were significantly higher from plots with the recently killed cover crop, but TP losses were not affected. Low-disturbance injection of swine manure into a standing cover crop can minimize plant damage and P losses in surface runoff while providing optimum P availability to a subsequent agronomic crop.

In situ dynamics of phosphorus in the rhizosphere solution of five species.

Root activity can modify the chemistry of the rhizosphere and alter phosphorus (P) availability and uptake. However, until recently, relatively little was known about the dynamics of soil solution P at the root surface because of our inability to measure in situ changes in solution P at the plant root. A mini-rhizotron experiment with corn (Zea mays L. cv. Stine 2250), soybean [Glycine max (L.) Merr. cv. Pioneer 3563), cottonwood (Populus deltoids L.), smooth brome (Bromus inermis Leyss.), and switchgrass (Panicum virgatum L.) was conducted to measure the spatial and temporal dynamics of P in the rhizosphere solution of a fine silty, P-rich calcareous soil (solid-phase total P concentration = 62 mg kg(-1), pH = 7.68) from western Iowa. Micro-suction cups were used to collect samples of soil solution from defined segments of the rhizosphere, and capillary electrophoresis (CE) was used to determine the P concentration of the soil solution. At the end of 10 d, a decreasing P concentration gradient in soil solution toward the root was observed in corn, cottonwood, and smooth brome. No clear rhizosphere effect was observed for soybean and switchgrass. Statistical analysis indicated significantly lower solution P concentrations in the rhizospheres of corn (p = 0.05), cottonwood (p = 0.01), and smooth brome (p = 0.01) compared with bulk soil solution. Results indicate that P depletion from rhizosphere soil solution depends on plant species. Under the conditions of this study, corn, cottonwood, and smooth brome were more effective in depleting solution P than soybean and switchgrass.

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reihardtii and Volvox carteri: phylogenetic origins and recent insertion of introns.

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble "proto-splice" sequences in the Chlamydomonas gene.

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.

Postpartum hypophagia in primiparous sows: I. Effects of gestation feeding level on feed intake, feeding behavior, and plasma metabolite concentrations during lactation.

To investigate the relationship between feeding level during gestation and voluntary feed intake, feeding behavior, and plasma metabolite levels during lactation, 18 crossbred, primiparous sows were assigned to two dietary treatments. From d 60 of gestation until farrowing, sows were fed either a standard level of feed (SL; 1.85 kg/d) or were allowed ad libitum (AL) access to feed. During a 28-d lactation all sows were allowed ad libitum access to feed. Eight SL and seven AL sows completed the experiment. Feed intake was measured daily during the final 40 d of gestation and throughout lactation. On d 105 of gestation and d 1, 7, 14, and 21 of lactation, feeding behavior was observed and blood samples were collected via indwelling catheters. Plasma was analyzed for nonesterified fatty acids (NEFA), insulin, glucose, and alpha-amino N. During gestation AL sows ate more feed than did SL sows. This increased intake was balanced by reduced feed intake during lactation, when AL sows ate less than SL sows (P < .001). The AL sows gained more weight during gestation (P < .001) and lost more weight during lactation (P < .005) than did the SL sows. Feed intake and weight change during gestation and lactation combined were not different between treatments (P = .85). During lactation, the AL sows ate fewer meals (P < .05) of a similar size than did the SL sows. During lactation, the area under the curve formed by plasma NEFA concentration was greater (P = .06) and that for insulin was less (P < .01) in AL than in SL sows.(ABSTRACT TRUNCATED AT 250 WORDS)

Postpartum hypophagia in primiparous sows: II. Effects of feeding level during gestation and exogenous insulin on lactation feed intake, glucose tolerance, and epinephrine-stimulated release of nonesterified fatty acids and glucose.

The objectives of this experiment were 1) to determine whether allowing sows ad libitum access to feed from d 60 of gestation affects glucose tolerance and 2) to determine whether exogenous insulin increases feed intake by preventing mobilization of nonesterified fatty acids (NEFA). Sixty crossbred sows were assigned to one of two feeding regimens during gestation, either a standard level of feed (SL; 1.85 kg/d) or allowed ad libitum access to feed (AL). Sows also received an injection of either .75 IU of insulin/kg BW or saline daily during the first 7 d of lactation. Exogenous insulin increased ADFI at d 7 of lactation (P = .07) and increased total feed intake at d 7 and 14 of lactation (P = .09). Total feed intake during d 0 to 21 was not affected by insulin treatment. Compared with the SL sows, the AL sows were less tolerant of glucose infusion (1 g of glucose/kg BW, i.v.) on d 1 of lactation (P < .01). Baseline and peak concentrations of insulin were not affected by feeding level during gestation (P = .4). Baseline and peak concentrations of NEFA were greater in AL sows than in SL sows (P < .001) and were not affected by insulin treatment (P = .39). Release of NEFA after epinephrine stimulation was greater in AL sows than in SL sows (P < .05). The data indicate that the reduced feed intake during lactation exhibited by sows that are overfed during gestation may be caused by insulin resistance. Exogenous insulin seems to increase feed intake by reducing plasma glucose rather than be affecting plasma NEFA.

Bioavailability of threonine in soybean meal for young pigs.

The bioavailability of threonine in solvent-extracted soybean meal for 10- to 20-kg pigs was determined using the slope-ratio method. In Exp. 1, the assay range was determined by feeding six diets to 144 pigs. The basal diet (.40% threonine) contained corn, soybean meal, and corn gluten meal. Five additional diets were formulated by supplementing the basal diet with .05 to .25% crystalline L-threonine in .05% increments. Weight gain, gain/feed, and plasma concentrations of threonine and urea responded quadratically (P < .05) to increasing dietary threonine. Breakpoints ranged from .51 to .54% dietary threonine. Experiment 2 consisted of seven trials in which a total of 239 pigs were used in a randomized complete block design. Pigs were penned individually and had ad libitum access to feed and water during the 21-d experiment. The same basal diet that was used in Exp. 1 was supplemented with .018, .053, or .070% threonine from either L-threonine or soybean meal. The weight gains of the pigs were partitioned to yield the response due to the supplemental threonine ingested. Multiple regression was performed on partitioned weight gain vs supplemental threonine intake, and the assay was tested for validity. The regression lines for L-threonine and soybean meal were linear (P < .05) and the intercepts were not significantly different (P > .10). The slope ratio for soybean meal:L-threonine was .80. Although the difference between the soybean meal and L-threonine slopes was not significant (P > .23), the best estimate of the bioavailability of threonine in soybean meal relative to that of L-threonine was 80%.

Prehospital care of acute myocardial infarction: a review.

Each year more than 1 million people in the United States suffer from acute myocardial infarction (MI) with most of the deaths occurring within hours of symptom onset. Over the last 25 years, different prehospital systems have evolved throughout the world which allow early cardiac monitoring and treatment of acute MI patients. Thrombolytic therapy in acute MI has been shown to decrease mortality and preserve left ventricular function when administered early after onset of symptoms. The potential role of Emergency Medical Services or Mobile Coronary Care Units in achieving early thrombolysis is under investigation. Several studies of prehospital interventions to achieve early thrombolysis are reviewed. The use of thrombolytics by prehospital personnel has been found to be feasible, safe, and effective in reducing time delays. However, whether this translates into clinical benefit remains to be seen.