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

A 5-Enolpyruvylshikimate 3-Phosphate Synthase Functions as a Transcriptional Repressor in Populus.

Long-lived perennial plants, with distinctive habits of inter-annual growth, defense, and physiology, are of great economic and ecological importance. However, some biological mechanisms resulting from genome duplication and functional divergence of genes in these systems remain poorly studied. Here, we discovered an association between a poplar (Populus trichocarpa) 5-enolpyruvylshikimate 3-phosphate synthase gene (PtrEPSP) and lignin biosynthesis. Functional characterization of PtrEPSP revealed that this isoform possesses a helix-turn-helix motif in the N terminus and can function as a transcriptional repressor that regulates expression of genes in the phenylpropanoid pathway in addition to performing its canonical biosynthesis function in the shikimate pathway. We demonstrated that this isoform can localize in the nucleus and specifically binds to the promoter and represses the expression of a SLEEPER-like transcriptional regulator, which itself specifically binds to the promoter and represses the expression of PtrMYB021 (known as MYB46 in Arabidopsis thaliana), a master regulator of the phenylpropanoid pathway and lignin biosynthesis. Analyses of overexpression and RNAi lines targeting PtrEPSP confirmed the predicted changes in PtrMYB021 expression patterns. These results demonstrate that PtrEPSP in its regulatory form and PtrhAT form a transcriptional hierarchy regulating phenylpropanoid pathway and lignin biosynthesis in Populus

Large-scale detector testing for the GAPS Si(Li) Tracker

Lithium-drifted silicon [Si(Li)] has been used for decades as an ionizing radiation detector in nuclear, particle, and astrophysical experiments, though such detectors have frequently been limited to small sizes (few cm$^2$) and cryogenic operating temperatures. The 10-cm-diameter Si(Li) detectors developed for the General Antiparticle Spectrometer (GAPS) balloon-borne dark matter experiment are novel particularly for their requirements of low cost, large sensitive area (~10 m$^2$ for the full 1440-detector array), high temperatures (near -40\,^\circC), and energy resolution below 4 keV FWHM for 20--100-keV x-rays. Previous works have discussed the manufacturing, passivation, and small-scale testing of prototype GAPS Si(Li) detectors. Here we show for the first time the results from detailed characterization of over 1100 flight detectors, illustrating the consistent intrinsic low-noise performance of a large sample of GAPS detectors. This work demonstrates the feasibility of large-area and low-cost Si(Li) detector arrays for next-generation astrophysics and nuclear physics applications.Comment: Updated to version accepted in IEEE Trans Nucl Sci. Minor changes to text, fixed plotting error on Fig. 5. Conclusions unchange

A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus

A greater understanding of biosynthesis, signaling and regulatory pathways involved in determining stem growth and secondary cell wall chemistry is important for enabling pathway engineering and genetic optimization of biomass properties. The present study describes a new functional role of PdIQD10, a Populus gene belonging to the IQ67-Domain1 family of IQD genes, in impacting biomass formation and chemistry. Expression studies showed that PdIQD10 has enhanced expression in developing xylem and tension-stressed tissues in Populus deltoides. Molecular dynamics simulation and yeast two-hybrid interaction experiments suggest interactions with two calmodulin proteins, CaM247 and CaM014, supporting the sequence-predicted functional role of the PdIQD10 as a calmodulin-binding protein. PdIQD10 was found to interact with specific Populus isoforms of the Kinesin Light Chain protein family, shown previously to function as microtubule-guided, cargo binding and delivery proteins in Arabidopsis. Subcellular localization studies showed that PdIQD10 localizes in the nucleus and plasma membrane regions. Promoter-binding assays suggest that a known master transcriptional regulator of secondary cell wall biosynthesis (PdWND1B) may be upstream of an HD-ZIP III gene that is in turn upstream of PdIQD10 gene in the transcriptional network. RNAi-mediated downregulation of PdIQD10 expression resulted in plants with altered biomass properties including higher cellulose, wall glucose content and greater biomass quantity. These results present evidence in support of a new functional role for an IQD gene family member, PdIQD10, in secondary cell wall biosynthesis and biomass formation in Populus

An investigation into customer perception and behaviour through social media research – an empirical study of the United Airline overbooking crisis

Airlines have been adopting yield management to optimise the perishable seat control problem and overbooking is a common strategy. This study outlines the connections between yield management, crises, and crisis communication. Using big data captured on a social media platform, this study aims to combine traditional yield management with emerging social big data analytics. As part of this, we use the twitter data on the 2017 United Airline (UA) to analyse the overbooking crisis. Our findings shed light on the importance of a more effective orchestration of yield management to avoid the escalation of crises during crisis communication phases