Lisa Bramer

Dynamics of the human gut microbiome in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by flares of inflammation with a periodic need for increased medication and sometimes even surgery. The aetiology of IBD is partly attributed to a deregulated immune response to gut microbiome dysbiosis. Cross-sectional studies have revealed microbial signatures for different IBD subtypes, including ulcerative colitis, colonic Crohns disease and ileal Crohns disease. Although IBD is dynamic, microbiome studies have primarily focused on single time points or a few individuals. Here, we dissect the long-term dynamic behaviour of the gut microbiome in IBD and differentiate this from normal variation. Microbiomes of IBD subjects fluctuate more than those of healthy individuals, based on deviation from a newly defined healthy plane (HP). Ileal Crohns disease subjects deviated most from the HP, especially subjects with surgical resection. Intriguingly, the microbiomes of some IBD subjects periodically visited the HP then deviated away from it. Inflammation was not directly correlated with distance to the healthy plane, but there was some correlation between observed dramatic fluctuations in the gut microbiome and intensified medication due to a flare of the disease. These results will help guide therapies that will redirect the gut microbiome towards a healthy state and maintain remission in IBD.

Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that, with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian Proteoform Quantification model (BP-Quant)1 that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern or the existence of multiple overexpressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab® and R packages.

Relative Influence of Trans-Pacific and Regional Atmospheric Transport of PAHs in the Pacific Northwest, U.S.

The relative influences of trans-Pacific and regional atmospheric transport on measured concentrations of polycyclic aromatic hydrocarbons (PAHs), PAH derivatives (nitro- (NPAH) and oxy-(OPAH)), organic carbon (OC), and particulate matter (PM) less than 2.5 μm in diameter (PM2.5) were investigated in the Pacific Northwest, U.S. in 2010–2011. Ambient high volume PM2.5 air samples were collected at two sites in the Pacific Northwest: (1.) Mount Bachelor Observatory (MBO) in the Oregon Cascade Range (2763 m above sea level (asl)) and 2.) Confederated Tribes of the Umatilla Indian Reservation (CTUIR) in the Columbia River Gorge (CRG) (954 m asl). At MBO, the 1,8-dinitropyrene concentration was significantly positively correlated with the time a sampled air mass spent over Asia, suggesting that this NPAH may be a good marker for trans-Pacific atmospheric transport. At CTUIR, NOx, CO2, and SO2 emissions from a 585 MW coal fired power plant, in Boardman OR, were found to be significantly positively correlated with PAH, OPAH, NPAH, OC, and PM2.5 concentrations. By comparing the Boardman Plant operational time frames when the plant was operating to when it was shut down, the plant was found to contribute a large percentage of the measured PAH (67%), NPAH (91%), OPAH (54%), PM2.5 (39%), and OC (38%) concentrations at CTUIR and the CRG prior to Spring 2011 and likely masked trans-Pacific atmospheric transport events to the CRG. Upgrades installed to the Boardman Plant in the spring of 2011 dramatically reduced the plant’s contribution to PAH and OPAH concentrations (by ∼72% and ∼40%, respectively) at CTUIR and the CRG, but not NPAH, PM2.5 or OC concentrations.

Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators.

Serum Proteome Profiles in Stricturing Crohn's Disease: A Pilot Study.

Background:Crohns disease (CD) is a form of inflammatory bowel disease with different described behaviors, including stricture. At present, there are no laboratory studies that can differentiate stricturing CD from other phenotypes of inflammatory bowel disease. We performed a pilot study to examine differences in the proteome among patients with stricturing CD, nonstricturing CD, and ulcerative colitis. Methods:Serum samples were selected from the Ocean State Crohns and Colitis Area Registry, an established cohort of patients with inflammatory bowel disease. Patients with CD with surgically resected stricture were matched with similar patients with CD without known stricture and with ulcerative colitis. Serum samples from each patient were digested and analyzed using liquid chromatography–mass spectrometry to characterize the proteome. Statistical analyses were performed to identify peptides and proteins that can differentiate CD with stricture. Results:Samples from 9 patients in each group (27 total patients) were analyzed. Baseline demographic characteristics were similar among the 3 groups. We quantified 7668 peptides and 897 proteins for analysis. Receiver operating characteristic analysis identified a subset of peptides with an area under the curve greater than 0.9, indicating greater separation potential. Partial least squares discriminant analysis was able to distinguish among the three groups with up to 70% accuracy by peptides and up to 80% accuracy by proteins. We identified the significantly different proteins and peptides and determined their function based on previously published literature. Conclusions:The serum of patients with stricturing CD, nonstricturing CD, and ulcerative colitis is distinguishable through proteomic analysis. Some of the proteins that differentiate the stricturing phenotype have been implicated in complement activation, fibrinolytic pathways, and lymphocyte adhesion.

Temporal profiles of plasma proteome during childhood development

Human blood plasma proteome reflects physiological changes associated with a childs development as well as development of disease states. While age-specific normative values are available for proteins routinely measured in clinical practice, there is paucity of comprehensive longitudinal data regarding changes in human plasma proteome during childhood. We applied TMT-10plex isobaric labeling-based quantitative proteomics to longitudinally profile the plasma proteome in 10 healthy children during their development, each with 9 serial time points from 9months to 15years of age. In total, 1828 protein groups were identified at peptide and protein level false discovery rate of 1% and with at least two razor and unique peptides. The longitudinal expression profiles of 1747 protein groups were statistically modeled and their temporal changes were categorized into 7 different patterns. The patterns and relative abundance of proteins obtained by LC-MS were also verified with ELISA. To our knowledge, this study represents the most comprehensive longitudinal profiling of human plasma proteome to date. The temporal profiles of plasma proteome obtained in this study provide a comprehensive resource and reference for biomarker studies in childhood diseases. Biological significance: A pediatric plasma proteome database with longitudinal expression patterns of 1747 proteins from neonate to adolescence was provided to the research community. 970 plasma proteins had age-dependent expression trends, which demonstrated the importance of longitudinal profiling study to identify the potential biomarkers specific to childhood diseases, and the requirement of strictly age-matched clinical samples in a cross-sectional study in pediatric population.

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

Blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n=11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n=10). To our knowledge, the current study represents the largest (>2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA. BIOLOGICAL SIGNIFICANCE The temporal changes of >2000 plasma proteins (at least quantified in two subjects), spanning the entire period of T1D natural progression were provided to the research community. Oxidative stress related proteins have consistently different dysregulated patterns in T1D group than in age-sex matched healthy controls, even prior to appearance of islet autoantibodies - the earliest sign of islet autoimmunity and pancreatic beta cell stress.

Dibenzo[def,p]chrysene transplacental carcinogenesis in wild‐type, Cyp1b1 knockout, and CYP1B1 humanized mice

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild‐type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb‐1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild‐type mice (P = 0.07). As with Cyp1b1 in wild‐type mice, constitutive hCYP1B1 protein is non‐detectable in liver but was induced with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. Wild‐type mice were 59% more likely to succumb to T‐cell Acute Lymphoblastic Leukemia (T‐ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755–762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60–69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild‐type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis.

Carbon Inputs From Riparian Vegetation Limit Oxidation of Physically Bound Organic Carbon Via Biochemical and Thermodynamic Processes

1 In light of increasing terrestrial carbon (C) transport across aquatic boundaries, the 2 mechanisms governing organic carbon (OC) oxidation along terrestrial-aquatic interfaces are 3 crucial to future climate predictions. Here, we investigate the biochemistry, metabolic pathways, 4 and thermodynamics corresponding to OC oxidation in the Columbia River corridor using ultra5 high resolution C characterization. We leverage natural vegetative differences to encompass 6 variation in terrestrial C inputs. Our results suggest that decreases in terrestrial C deposition 7 associated with diminished riparian vegetation induce oxidation of physically -bound OC. We 8 also find that contrasting metabolic pathways oxidize OC in the presence and absence of 9 vegetation and—in direct conflict with the ‘priming’ concept—that inputs of water-soluble and 10 thermodynamically favorable terrestrial OC protects bound-OC from oxidation. In both 11 environments, the most thermodynamically favorable compounds appear to be preferentially 12 oxidized regardless of which OC pool microbiomes metabolize. In turn, we suggest that the 13 extent of riparian vegetation causes sediment microbiomes to locally adapt to oxidize a particular 14 pool of OC, but that common thermodynamic principles govern the oxidation of each pool (i.e., 15 water-soluble or physically-bound). Finally, we propose a mechanistic conceptualization of OC 16 oxidation along terrestrial-aquatic interfaces that can be used to model heterogeneous patterns of 17 OC loss under changing land cover distributions. 18 19 20 21 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/105486 doi: bioRxiv preprint first posted online Feb. 2, 2017;

Dispersal limitation and thermodynamic constraints govern spatial structure of permafrost microbial communities

Understanding drivers of permafrost microbial community composition is critical for understanding permafrost microbiology and predicting ecosystem responses to thaw. We hypothesize that permafrost communities are shaped by physical constraints imposed by prolonged freezing, and exhibit spatial distributions that reflect dispersal limitation and selective pressures associated with these physical constraints. To test this, we characterized patterns of environmental variation and microbial community composition in permafrost across an Alaskan boreal forest landscape. We used null modeling to estimate the importance of selective and neutral assembly processes on community composition, and identified environmental factors influencing ecological selection through regression and structural equation modeling (SEM). Proportionally, the strongest process influencing community composition was dispersal limitation (0.36), exceeding the influence of homogenous selection (0.21), variable selection (0.16) and homogenizing dispersal (0.05). Fe(II) content was the most important factor explaining variable selection, and was significantly associated with total selection by univariate regression (R2 = 0.14, P = 0.003). SEM supported a model in which Fe(II) content mediated influences of the Gibbs free energy of the organic matter pool and organic acid concentration on total selection. These findings suggest that the dominant processes shaping microbial communities in permafrost result from the stability of the permafrost environment, which imposes dispersal and thermodynamic constraints.