Application of Systems Biology Analysis to Hepatic Injury Following Hemorrhagic Shock

University of Minnesota Ph.D. dissertation. May 2014. Major: Integrated Biosciences. Advisor: Dr. Gregory J. Beilman. 1 PDF (ix, 151 pages) + 1 zipped file containing 8 charts in Excel format.Introduction: This dissertation is focused on the metabolomic and transcriptomic changes that occur as a result of carbohydrate prefeeding during hemorrhagic shock and trauma within the liver of a porcine model. The risk of trauma and hemorrhagic shock continues to be an important issue in both military and civilian sectors. As such, we explored the impact of a prior fed state upon the overall response to hemorrhagic shock and resuscitation. The primary hypotheses were that changes in metabolism at the metabolomic and transcriptomic levels would be dependent upon the fed state. In addition, this thesis explores a more comprehensive analysis of metabolomics datasets to standardize analysis and improve overall consistency.Materials and Methods: Algorithm comparison was accomplished using six commonly applied methods to three synthetic datasets, of different sample sizes, and three openly accessible published datasets. This comparison also incorporated metrics to measure consistency of identified features (i.e. stability) to provide further confidence in results. Metabolomics analysis was accomplished with nuclear magnetic resonance spectroscopy (NMR) and Chenomx software to profile and quantify metabolites in liver extracts. The metabolome was subsequently analyzed with partial least squares discriminant analysis (PLS-DA). Transcriptomics analysis was conducted using next-generation sequencing (NGS) technology to employ RNA-sequencing (RNA-seq) on mRNA extracts from liver biopsies. The RNA-seq data was analyzed using typical processing techniques to generate a count matrix and subsequently analyzed with the Bioconductor package EdgeR. Results: The comparison of algorithms showed that the best algorithm is associated with differently structured datasets (e.g. number of features, number of groups, sample size, etc.). Analysis of the liver metabolome revealed changes in carbon energy sources, amino acid metabolism, oxidative stress, and membrane maintenance. Transcriptomic analysis revealed changes in carbohydrate metabolism, cytokine inflammation, cholesterol synthesis and apoptosis. In addition, there is evidence of increased cytoskeleton reorganization which may correspond to a shrunken, catabolic state which provides and anti-inflammatory condition to mitigate cellular damage.Conclusion: The response to hemorrhagic shock and resuscitation is altered with respect to a fasted or carbohydrate prefed state. Metabolomics and transcriptomic analyses suggest altered metabolic pathways as a result of fed state. Altered carbohydrate metabolism was readily identified thereby confirming both methods were successful. Additionally, indications of membrane maintenance that follow cytoskeletal remodeling and cellular shrinkage are potentially reflected by 3-Hydroxyisovalerate and sn-Glycero-3-phosphocholine. These results provide further evidence for pre-conditioning (e.g. altered diet) and hypertonic resuscitation methods to possibly improve patient outcome. Further research is required in alternative prefeeding substrates (e.g. protein, lipid, etc.) as well as improving the integration of different systems level datasets to understand more thoroughly the systemic effects of hemorrhagic shock and resuscitation

Design of a Protective Single-Dose Intranasal Nanoparticle-Based Vaccine Platform for Respiratory Infectious Diseases

Despite the successes provided by vaccination, many challenges still exist with respect to controlling new and re-emerging infectious diseases. Innovative vaccine platforms composed of adaptable adjuvants able to appropriately modulate immune responses, induce long-lived immunity in a single dose, and deliver immunogens in a safe and stable manner via multiple routes of administration are needed. This work describes the development of a novel biodegradable polyanhydride nanoparticle-based vaccine platform administered as a single intranasal dose that induced long-lived protective immunity against respiratory disease caused by Yesinia pestis, the causative agent of pneumonic plague. Relative to the responses induced by the recombinant protein F1-V alone and MPLA-adjuvanted F1-V, the nanoparticle-based vaccination regimen induced an immune response that was characterized by high titer and high avidity IgG1 anti-F1-V antibody that persisted for at least 23 weeks post-vaccination. After challenge, no Y. pestis were recovered from the lungs, livers, or spleens of mice vaccinated with the nanoparticle-based formulation and histopathological appearance of lung, liver, and splenic tissues from these mice post-vaccination was remarkably similar to uninfected control mice

New town : a synthesis by re-vision

M.S.H. Randal Roar

Fed State Prior to Hemorrhagic Shock and Polytrauma in a Porcine Model Results in Altered Liver Transcriptomic Response

<div><p>Hemorrhagic shock is a leading cause of trauma-related mortality in both civilian and military settings. Resuscitation often results in reperfusion injury and survivors are susceptible to developing multiple organ failure (MOF). The impact of fed state on the overall response to shock and resuscitation has been explored in some murine models but few clinically relevant large animal models. We have previously used metabolomics to establish that the fed state results in a different metabolic response in the porcine liver following hemorrhagic shock and resuscitation. In this study, we used our clinically relevant model of hemorrhagic shock and polytrauma and the Illumina HiSeq platform to determine if the liver transcriptomic response is also altered with respect to fed state. Functional analysis of the response to shock and resuscitation confirmed several typical responses including carbohydrate metabolism, cytokine inflammation, decreased cholesterol synthesis, and apoptosis. Our findings also suggest that the fasting state, relative to a carbohydrate prefed state, displays decreased carbohydrate metabolism, increased cytoskeleton reorganization and decreased inflammation in response to hemorrhagic shock and reperfusion. Evidence suggests that this is a consequence of a shrunken, catabolic state of the liver cells which provides an anti-inflammatory condition that partially mitigates hepatocellar damage.</p></div

PLS-DA scores plots for the S45-B time interval.

<p>PLS-DA scores plots show model discrimination between FS and CPF animals during the response to shock (S45-B) in each of the four compartments (liver, muscle, serum, urine). Models are of varying quality and statistical significance as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124467#pone.0124467.t001" target="_blank">Table 1</a> but indicate that there is a difference in response to shock according to feeding status.</p

Purine degradation/salvage pathway.

<p>Metabolomics evidence from four compartments suggests alternate routes for observed differences in purine abundances. ATP is degraded in a series of reactions to uric acid or allantoin. Under non-stress conditions, this degradation process proceeds at a low level. When physiologic conditions change, intermediates in the pathway can be diverted to meet these demands. For example, during fasting, IMP can be salvaged for the production of ATP thus reducing the level of HX. In our study, HX levels are higher at baseline in liver of CPF animals. We propose that this observation is a result of increased salvage in FS animals. During ischemia 5'-nucleotidase acts on AMP to generate adenosine, a potential vasodilator. In our study, adenosine levels are ~3X higher in the liver of CPF animals when compared to FS animals suggesting a greater need for vasodilation. Metabolites in red were identified as VIP metabolites by PLS-DA analysis. Letters in superscript indicate the compartment the metabolite was observed in: liver (L), muscle (M), serum (S), or urine (U).</p

Metabolic profile associated with feeding status at Baseline.

<p>a) Higher levels of glucose are observed in liver, muscle, and serum of CPF animals. Urine choline and betaine as well as a reduced level of muscle ATP support the hypothesis of enhanced glucose-associated biosynthesis. Formation of a mitochondrial megachannel that allows for exit of citric acid cycle intermediates is suggested by elevated levels of fumarate, succinate, and citrate in the urine of CPF animals. b) Levels of liver, muscle, and serum BCAAs (isoleucine, leucine, and valine) as well as serum AA (tyrosine, serine, and threonine) are all higher in fasted animals at baseline suggesting increased proteolysis. The difference in the levels of serum urea (#) approach significance (p = 0.09) further supporting this hypothesis. * = VIP metabolite where levels observed in CPF animals > than that observed in FS animals. ◆ = VIP metabolite where levels observed in FS animals > than that observed in CPF animals. NV = metabolite not visible in that compartment.</p

Graphical representation of the experimental timeline described in Methods.

<p>Graphical representation of the experimental timeline described in Methods.</p

Metabolic profile associated with the response to shock.

<p>Regardless of pre-trauma dietary state, both CPF and FS animals exhibit a similar metabolic response to shock. Glucose levels increase from baseline in the liver, muscle, and serum. This is attributed to the breakdown of glycogen. The increase is greater in CPF animals presumably due to the enhanced glycogen stores in CPF animals. Both tissues also exhibit increased levels of BCAA (isoleucine, leucine, valine), suggesting more proteolysis during shock than at baseline. Greater increases in BCAA levels are observed in FS animals. Muscle creatine phosphate (PCr) levels decrease in both groups during shock compared to baseline but the decrease is greater by almost 4-fold in FS animals when compared to CPF animals. Since muscle ATP levels are not differentiating between the two groups, FS animals appeared to rely more heavily on this non-oxidative mode of ATP generation. Alanine levels (^) increase to a greater extent in the muscle of FS animals. This difference could reflect the time lag necessary to shift the metabolic machinery dedicated to glucose use in CPF animals at baseline to that necessary to process the greater load of amino acids at shock. The urine metabolome is not profiled at this time interval. During shock, animals minimize fluid loss and metabolite levels would not accurately reflect metabolic activities. * = VIP metabolite where levels observed in CPF animals > than that observed in FS animals. ◆ = VIP metabolite where levels observed in FS animals > than that observed in CPF animals.</p

Heatmap of log2 fold changes of genes associated with cytokine related genes in carbohydrate prefed (CPF) animals at each resuscitation timepoint (2, 8, and 20 hours) relative to Baseline (B).

<p>Rows are differentially expressed genes (DEGs) following RNA sequencing. Columns denote the respective timepoints Baseline (B), 2 hours full resuscitation change from Baseline (FR2vB), 8 hours full resuscitation change from Baseline (FR8vB), and 20 hours full resuscitation change from Baseline (FR20vB). Green denotes increased mRNA expression with respect to Baseline whereas red denotes the opposite.</p