A Four-Compartment Metabolomics Analysis of the Liver,Muscle, Serum,and Urine Response to Polytrauma with Hemorrhagic Shock following Carbohydrate Prefeed

Objective

Hemorrhagic shock accompanied by injury represents a major physiologic stress. Fasted animals are often used to study hemorrhagic shock (with injury). A fasted state is not guaranteed in the general human population. The objective of this study was to determine if fed animals would exhibit a different metabolic profile in response to hemorrhagic shock with trauma when compared to fasted animals.

Methods

Proton (1H) NMR spectroscopy was used to determine concentrations of metabolites from four different compartments (liver, muscle, serum, urine) taken at defined time points throughout shock/injury and resuscitation. PLS-DA was performed and VIP lists established for baseline, shock and resuscitation (10 metabolites for each compartment at each time interval) on metabolomics data from surviving animals.

Results

Fed status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The death rate for CPF animals is higher than FS animals (47 vs 28%). The majority of deaths occur post-resuscitation suggesting reperfusion injury. The metabolomics response to shock reflects priorities evident at baseline. FS animals raise the baseline degree of proteolysis to provide additional amino acids for energy production while CPF animals rely on both glucose and, to a lesser extent, amino acids. During early resuscitation levels of metabolites associated with energy production drop, suggesting diminished demand.

Conclusions

Feeding status prior to the occurrence of hemorrhagic shock with injury alters the metabolic course of this trauma and potentially affects mortality. The response to shock reflects metabolic priorities at baseline.     

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.     

Comparing land surface phenology derived from satellite and GPS network microwave remote sensing

The land surface phenology (LSP) start of season (SOS) metric signals the seasonal onset of vegetation activity, including canopy growth and associated increases in land-atmosphere water, energy and carbon (CO₂) exchanges influencing weather and climate variability. The vegetation optical depth (VOD) parameter determined from satellite passive microwave remote sensing provides for global LSP monitoring that is sensitive to changes in vegetation canopy water content and biomass, and insensitive to atmosphere and solar illumination constraints. Direct field measures of canopy water content and biomass changes desired for LSP validation are generally lacking due to the prohibitive costs of maintaining regional monitoring networks. Alternatively, a normalized microwave reflectance index (NMRI) derived from GPS base station measurements is sensitive to daily vegetation water content changes and may provide for effective microwave LSP validation. We compared multiyear (2007–2011) NMRI and satellite VOD records at over 300 GPS sites in North America, and their derived SOS metrics for a subset of 24 homogenous land cover sites to investigate VOD and NMRI correspondence, and potential NMRI utility for LSP validation. Significant correlations (P?<?0.05) were found at 276 of 305 sites (90.5 %), with generally favorable correspondence in the resulting SOS metrics (r ²?=?0.73, P?<?0.001, RMSE = 36.8 days). This study is the first attempt to compare satellite microwave LSP metrics to a GPS network derived reflectance index and highlights both the utility and limitations of the NMRI data for LSP validation, including spatial scale discrepancies between local NMRI measurements and relatively coarse satellite VOD retrievals.     

The PhoU Protein from Escherichia coli Interacts with PhoR,PstB, and Metals To Form a Phosphate-Signaling Complex at the Membrane

Robust growth in many bacteria is dependent upon proper regulation of the adaptive response to phosphate (P_i) limitation. This response enables cells to acquire P_i with high affinity and utilize alternate phosphorous sources. The molecular mechanisms of P_i signal transduction are not completely understood. PhoU, along with the high-affinity, P_i-specific ATP-binding cassette transporter PstSCAB and the two-component proteins PhoR and PhoB, is absolutely required for P_i signaling in Escherichia coli. Little is known about the role of PhoU and its function in regulation. We have demonstrated using bacterial two-hybrid analysis and confirmatory coelution experiments that PhoU interacts with PhoR through its PAS (Per-ARNT-Sim) domain and that it also interacts with PstB, the cytoplasmic component of the transporter. We have also shown that the soluble form of PhoU is a dimer that binds manganese and magnesium. Alteration of highly conserved residues in PhoU by site-directed mutagenesis shows that these sites play a role in binding metals. Analysis of these phoU mutants suggests that metal binding may be important for PhoU membrane interactions. Taken together, these results support the hypothesis that PhoU is involved in the formation of a signaling complex at the cytoplasmic membrane that responds to environmental P_i levels.     

Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.     

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC₅₀ values as low as 19 nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFα production in peripheral human monocytes.     

Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA degradation over time in wet environments, and have not been performed on fecal pellets of ungulates. Therefore, our objective was to determine the length of time a fecal pellet from a Sitka black-tailed deer (Odocoileus hemionus sitkensis) could remain in the field in a temperate rainforest environment before the DNA became too degraded for individual identification. Pellets were extracted from the rectum of recently killed deer and placed in an environment protected from rainfall and in an environment exposed to rainfall. Pellets from each treatment group were sampled at intervals of 2, 7, 14, 21, and 28 days after deer harvest. DNA was extracted from sampled pellets and individual samples were genotyped using microsatellite markers. Amplification failure and errors (dropout and false alleles) were recorded to determine extent of DNA degradation. Eighty percent of samples in the protected environment and 22% of samples in the exposed environment were successfully genotyped during the 28-day experiment. With no samples being successfully genotyped in the exposed environment after 7 days, our study showed that rainfall significantly increases degradation rates of DNA from ungulate pellets.     

Soil carbon and nitrogen mineralization following deposition of insect frass and greenfall from forests under elevated CO2 and O3

Elevated CO₂ and O₃ alter tree quality and the quality of herbivore inputs, such as frass, to forest soil. Altered quality or quantity of herbivore inputs to the forest floor can have large impacts on belowground processes. We collected green leaves and frass from whitemarked tussock moth caterpillars from aspen-birch stands at the Aspen Free Air CO₂ Enrichment (FACE) site near Rhinelander, WI, USA. Small or large quantities of frass, greenfall, or a 1:1 ratio of frass and greenfall were added to microcosms for each FACE treatment (control, +CO₂, +O₃, +CO₂+O₃). We measured initial frass and greenfall quality, and recorded microbial respiration, and nitrate leaching over 40 days. Elevated carbon dioxide (eCO₂) and tropospheric ozone (eO₃) significantly altered the carbon, nitrogen, and condensed tannin content of insect frass and green leaves. Although FACE treatments affected input quality, they had minimal effect on microbial respiration and no effect on nitrogen leaching. In contrast, input quantity substantially influenced microbial respiration and nitrate leaching. Respiratory carbon loss and nitrate immobilization were nearly double in microcosms receiving large amounts of herbivore inputs than those receiving no herbivore inputs. Small amounts of herbivore inputs, however, did not significantly alter microbial respiration or immobilization, suggesting that effects of herbivore inputs on soil processes will be detected only at moderate to high herbivory/input levels. These results suggest that subtle changes in frass and greenfall quality may not affect soil nutrient cycling. In contrast, environmental change induced increases in insect population size or frass and greenfall inputs to the soil may substantially impact nutrient cycling.