The role of manganese peroxidase in biomass conversion technologies

Currently, the technologies used to separate lignocellulosic biomass into its component parts (cellulose, hemicellulose, and lignin) and enzymatically hydrolyze the cellulose to glucose for conversion to ethanol could be improved economically and in terms of efficiency. A major impediment to utilizing the biomass is the presence of residual lignin. The residual lignin "poisons" the cellulase enzymes and as a result, a lower glucose yield is obtained. A higher titer of cellulases could be used to increase the yield of glucose, but cellulases are expensive. A possibility to reduce the cost of cellulase enzymes and improve the efficiency, economics, extent and/or rate of hydrolysis is to use lignin-degrading fungal heme peroxidase such as manganese peroxidases. An efficient and economical glucose assay was developed to monitor the rate and extent of enzymatic hydrolysis of cellulose. The glucose assay is based on the glucose oxidase horseradish peroxidase enzymatic method, but uses bulk enzymes making it more economical than commercial kits. The glucose assay also has a higher throughput rate and is more economical than HPLC. In order to evaluate possible synergistic effects between cellulases and manganese peroxidase (MnP), rMnP was produced in high cell density fed-batch cultivations by the genetically engineered yeast, P. pastoris. In addition, a mathematic model was developed to describe the temperature dependant growth of P. pastoris and consumption of glucose and the production and temperature dependent degradation of rMnP in the bioreactor broth. The model successfully predicted the cell growth, substrate consumption, and rMnP production for the base case and also for cultivations with varying fed-batch air flow rates (k[subscript L]a) and temperatures. The production of rMnP requires cultures amended with exogenous heme and there are several sources of heme. Through shake flask experiments and bioreactor cultivations it was determined that 0.1 g/L of heme was necessary for producing high titers of rMnP (4,500 U/L). It was also determined that not all types of heme will yield the same rMnP titer. The water soluble fraction post pretreatment of lignocellulosic biomass contains inhibitors to fermentation such as 5-hydroxymethyl furfural (HMF) and furfural. rMnP was shown to degrade HMF (1 g/L) and furfural (1 g/L) and detoxify medium containing these inhibitors. The rMnP reduced furfural and HMF, measured by absorbance at 276 and 286 nm respectively and the degree of absorbance decrease was dependent on rMnP concentration. Furfural was more readily degraded by rMnP than HMF. Growth assays using S. cerevisiae indicated rMnP treatment detoxified medium containing furfural and HMF. The optimal conditions (temperature, pH, and buffer) for enzyme activity were determined for both AccelleraseTM 1000 (commercially available combination of cellulases) and rMnP using filter paper as a substrate. Woody biomass and corn stover were pretreated and then exposed to simultaneous or sequential treatment with rMnP and AccelleraseTM 1000. The results for the sequential treatment of Ponderosa pine and corn stover with rMnP and AccelleraseTM 1000 was inconclusive as to whether or not rMnP effected the production of glucose due to the high variability between replicates. Finally, part of my doctoral program was significant mentoring and facilitation of undergraduate research. A significant portion of my time was dedicated toward senior laboratory teaching assistance, serving as a mentor for high school students, and participating in research mentoring with 24 undergraduate students over five years, 19 of whom were women

The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes.

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance. Using cultured 3T3-L1 adipocytes, we established a model of physiologically-derived oxidative stress by inhibiting the cycling of glutathione and thioredoxin, which induced insulin resistance as measured by impaired insulin-stimulated 2-deoxyglucose uptake. Using time-resolved transcriptomics, we found > 2000 genes differentially-expressed over 24 hours, with specific metabolic and signalling pathways enriched at different times. We explored this coordination using a knowledge-based hierarchical-clustering approach to generate a temporal transcriptional cascade and identify key transcription factors responding to oxidative stress. This response shared many similarities with changes observed in distinct insulin resistance models. However, an anti-oxidant reversed insulin resistance phenotypically but not transcriptionally, implying that the transcriptional response to oxidative stress is insufficient for insulin resistance. This suggests that the primary site by which oxidative stress impairs insulin action occurs post-transcriptionally, warranting a multi-level 'trans-omic' approach when studying time-resolved responses to cellular perturbations

Role of dietary fatty acids in mammary gland development and breast cancer

Breast cancer is the most common cancer among women worldwide. Estimates suggest up to 35% of cases may be preventable through diet and lifestyle modification. Growing research on the role of fats in human health suggests that early exposure in life to specific fatty acids, when tissues are particularly sensitive to their environment, can have long-term health impacts. The present review examines the role of dietary fat in mammary gland development and breast cancer throughout the lifecycle. Overall, n-3 polyunsaturated fatty acids have promising cancer-preventive effects when introduced early in life, and warrant further research to elucidate the mechanisms of action

Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis

Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Using proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered on IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with Bruton’s tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) pathway activation as a central signal transduction network in HS. These data provide preclinical evidence to accelerate the path toward clinical trials targeting BTK and SYK signaling in moderate-to-severe HS

The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes.

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance. Using cultured 3T3-L1 adipocytes, we established a model of physiologically-derived oxidative stress by inhibiting the cycling of glutathione and thioredoxin, which induced insulin resistance as measured by impaired insulin-stimulated 2-deoxyglucose uptake. Using time-resolved transcriptomics, we found > 2000 genes differentially-expressed over 24 hours, with specific metabolic and signalling pathways enriched at different times. We explored this coordination using a knowledge-based hierarchical-clustering approach to generate a temporal transcriptional cascade and identify key transcription factors responding to oxidative stress. This response shared many similarities with changes observed in distinct insulin resistance models. However, an anti-oxidant reversed insulin resistance phenotypically but not transcriptionally, implying that the transcriptional response to oxidative stress is insufficient for insulin resistance. This suggests that the primary site by which oxidative stress impairs insulin action occurs post-transcriptionally, warranting a multi-level 'trans-omic' approach when studying time-resolved responses to cellular perturbations

BOLD Imaging in Awake Wild-Type and Mu-Opioid Receptor Knock-Out Mice Reveals On-Target Activation Maps in Response to Oxycodone

Blood oxygen level dependent (BOLD) imaging in awake mice was used to identify differences in brain activity between wild-type, and Mu (µ) opioid receptor knock-outs (MuKO) in response to oxycodone (OXY). Using a segmented, annotated MRI mouse atlas and computational analysis, patterns of integrated positive and negative BOLD activity were identified across 122 brain areas. The pattern of positive BOLD showed enhanced activation across the brain in WT mice within 15 min of intraperitoneal administration of 2.5 mg of OXY. BOLD activation was detected in 72 regions out of 122, and was most prominent in areas of high µ opioid receptor density (thalamus, ventral tegmental area, substantia nigra, caudate putamen, basal amygdala and hypothalamus), and focus on pain circuits indicated strong activation in major pain processing centers (central amygdala, solitary tract, parabrachial area, insular cortex, gigantocellularis area, ventral thalamus primary sensory cortex and prelimbic cortex). Importantly, the OXY-induced positive BOLD was eliminated in MuKO mice in most regions, with few exceptions (some cerebellar nuclei, CA3 of the hippocampus, medial amygdala and preoptic areas). This result indicates that most effects of OXY on positive BOLD are mediated by the µ opioid receptor (on-target effects). OXY also caused an increase in negative BOLD in WT mice in few regions (16 out of 122) and, unlike the positive BOLD response the negative BOLD was only partially eliminated in the MuKO mice (cerebellum), and in some case intensified (hippocampus). Negative BOLD analysis therefore shows activation and deactivation events in the absence of the µ receptor for some areas where receptor expression is normally extremely low or absent (off-target effects). Together, our approach permits establishing opioid-induced BOLD activation maps in awake mice. In addition, comparison of WT and MuKO mutant mice reveals both on-target and off-target activation events, and set an OXY brain signature that should, in the future, be compared to other µ opioid agonists