Xylose isomerase from Piromyces:Characterization and engineering for improving S. cerevisiae-based lignocellulosic bioethanol production

Lignocellulosic bioethanol has been recognized as a possible fossil fuel alternative. This so-called “second-generation” bioethanol has gained growing interest as it can be produced using agricultural waste material or side products in the food industry as opposed to “first-generation” bioethanol which is produced from food sources such as sugar canes or corn. While the fermentation processes for first-generation bioethanol are well-established, second-generation bioethanol processes are more laborious due to the recalcitrant structural properties of lignocellulose. The process includes pretreatment of biomass, hydrolysis, fermentation as well as distillation and optimizations are required in all steps. One of the optimizations for cost-effective lignocellulosic bioethanol relies on the maximal utilization of the biomass. Following glucose which is the most abundant fermentable sugar in the biomass, xylose is the second most abundant sugar. Therefore, efficient fermentation of xylose is critical. However, S. cerevisiae, the commonly used organism for the fermentation process is inherently incapable of growing on xylose. This problem is solved by incorporating a heterologous xylose isomerase which converts xylose to xylulose which can then naturally be further assimilated by S. cerevisiae producing ethanol. Although the use of xylose isomerase enables the xylose fermentation of S. cerevisiae, its activity is yet sub-optimal and therefore requires improvement. This thesis describes biochemical and structural characterization of xylose isomerase from fungal strain Piromyces. Using this information, several enzyme engineering approaches were explored which resulted in discovering improved variants. The different approaches used, properties of discovered variants and their effects on xylose fermentation are explained throughout the thesis

Calculus Instructors' Resources, Orientations, and Goals in Teaching Low-Achieving Students

Teaching and learning calculus has been the subject of mathematics education research for many years. Although the literature is mainly concerned with students’ difficulties with calculus, research on mathematicians’ day to day activities is still scarce. Using Schoenfeld’s Resources, Orientations and Goals (ROGs) framework, this study examined four instructors’ ROGs in teaching calculus to low-achieving students. The findings revealed the in depth pedagogical experiences of mathematicians and their deliberation in helping students. The study suggested that building research based models and frameworks results in richer studies that would be more beneficial to both students and instructors

Development of Small Molecules as Chemical Probes for Understanding the Pathology of Alzheimer???s Disease

Department of ChemistryAlzheimer???s Disease (AD), responsible for most dementia cases, is one of the prevalent neurodegenerative diseases. Multiple features (e.g., neuroinflammation, accumulation of amyloid aggregates, dyshomeostasis of metal ions) have been implicated in the pathogenesis of AD, and such multifactorial nature impedes the investigation of the effective treatment of the disease. To elucidate the unrevealed biological actions linked with the pathogenesis and identify the remedy for the disease, many studies have been focused on devising and utilizing small molecules as chemical probes or potential therapeutics. In Chapter 1, we describe the development of a small molecule, 1, as a chemical agent for understanding the interconnection between neuroinflammation and amyloid-?? (A??) peptides as well as a potential drug candidate against AD. By intervening in the inflammatory process of microglia, 1 could promote the microglial phagocytosis of A?? and subsequently ameliorates cognitive defects in vivo. These results could provide an advanced insight to future discovery of effective anti-neuroinflammatory drugs against neurodegeneration. In Chapter 2, a turn-on near-infrared (near-IR) fluorescent probe, 2, for imaging A?? aggregates is introduced. The ability of 2 was evaluated to alter fluorescent response depending on the degree of A?? aggregation, at the near-IR region, and its suitability for imaging A?? aggregates under cellular environments. This work allows us to attain new perceptions to devise improved fluorescent probes with better specificity and bioapplicability, via further structural modifications. In Chapter 3, the design of small molecules capable of detecting metal ions in living cells is described. These studies present the capability of our molecules to monitor metal ions in living cells. Overall, our studies illustrate the utilization of small molecules to gain a better understanding of the pathogenic factors of interest, which will contribute to invention of chemical agents used for elucidating the pathology of AD.clos

Flavin-Tag:A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo-and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-Tag. We show that this peptide can be as short as 7 residues and can be located at the N-Terminus, C-Terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro, rendering the "Flavin-Tag"method a powerful tool for the selective decoration of proteins with a biocompatible redox-Active fluorescent chromophore

Measurements of Location-Dependent Nitric Oxide Levels on Skin Surface in relation to Acupuncture Point

Location-dependent skin surface’s partial nitric oxide pressure (pNO) is studied using highly sensitive amperometric NO microsensor with a small sensing area (diameter  = 76 μm). The pNO level of LI4 (Hegu) acupuncture point is measured and compared with the pNO level of nonacupuncture point. In addition, the mapping of pNO is carried out over the left wrist skin area one- as well as two-dimensionally. Statistically higher pNO levels near the position of acupuncture points than non-acupuncture points are observed consistently, implying tight relationship between the level of NO release of skin and acupuncture points. The amperometric planar NO microsensor successfully monitors the heterogeneity of skin pNO distribution in high spatial resolution due to its advantageous features such as high sensitivity and small sensing dimension. The current study suggests the direct connection between NO and acupuncture points and possibly provides beneficial information to understand physiological roles and basis of the acupuncture points

Q-HyViT: Post-Training Quantization for Hybrid Vision Transformer with Bridge Block Reconstruction

Recently, vision transformers (ViTs) have superseded convolutional neural networks in numerous applications, including classification, detection, and segmentation. However, the high computational requirements of ViTs hinder their widespread implementation. To address this issue, researchers have proposed efficient hybrid transformer architectures that combine convolutional and transformer layers with optimized attention computation of linear complexity. Additionally, post-training quantization has been proposed as a means of mitigating computational demands. For mobile devices, achieving optimal acceleration for ViTs necessitates the strategic integration of quantization techniques and efficient hybrid transformer structures. However, no prior investigation has applied quantization to efficient hybrid transformers. In this paper, we discover that applying existing PTQ methods for ViTs to efficient hybrid transformers leads to a drastic accuracy drop, attributed to the four following challenges: (i) highly dynamic ranges, (ii) zero-point overflow, (iii) diverse normalization, and (iv) limited model parameters ($<$5M). To overcome these challenges, we propose a new post-training quantization method, which is the first to quantize efficient hybrid ViTs (MobileViTv1, MobileViTv2, Mobile-Former, EfficientFormerV1, EfficientFormerV2) with a significant margin (an average improvement of 8.32\% for 8-bit and 26.02\% for 6-bit) compared to existing PTQ methods (EasyQuant, FQ-ViT, and PTQ4ViT). We plan to release our code at \url{https://github.com/Q-HyViT}.Comment: 12 pages, 8 figure

Prevalent Multidrug-resistant Nonvaccine Serotypes in Pneumococcal Carriage of Healthy Korean Children Associated with the Low Coverage of the Seven-valent Pneumococcal Conjugate Vaccine

AbstractObjectivesOur previous longitudinal multicenter-based carriage study showed that the average carriage rate of Streptococcus pneumoniae was 16.8% in 582 healthy children attending kindergarten or elementary school in Seoul, Korea. We assessed serotype-specific prevalence and antimicrobial resistance among colonizing pneumococcal isolates from young children in the era of low use of the seven-valent pneumococcal conjugate vaccine (PCV7).MethodsSerotypes were determined by an agglutination test with specific antisera or by a multiplex polymerase chain reaction (PCR) assay. An antimicrobial susceptibility test was performed with broth microdilution in Korean 96-well panels from Dade-MicroScan (Sacramento, CA, USA).ResultsPneumococcal colonization patterns were dynamic and longterm persistent carriage was rare, which indicated a sequential turnover of pneumococcal strains. Of the 369 pneumococci (except for 23 killed isolates), 129 (34.9%) isolates were PCV7 vaccine serotypes (VTs); 213 (57.8%) isolates were nonvaccine serotypes (NVTs); and the remaining 27 (7.2%) isolates were nontypable (NT). The highest rates of multidrug resistance (MDR) were observed in VTs (86.0%; 111/129 isolates) and NVTs (70.0%; 149/213 isolates).ConclusionThis study overall showed the frequent carriage of VTs and NVTs with MDR in healthy children attending kindergarten or elementary school. Efforts should be directed toward reducing the extensive prescription of antibiotics and using new broader vaccines to reduce the expansion of MDR strains of NVTs in our community

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

[Image: see text] Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5′-phosphate) can functionally substitute the natural deazaflavin F(420) and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae. Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F(420) yield of native F(420)-producing organisms such as Mycobacterium smegmatis, but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L(–1) h(–1) and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae, in vivo FOP production by SpRFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a SpRFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

Background: Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results: We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion: This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism

Content validity and test-retest reliability of patient perception of intensity of urgency scale (PPIUS) for overactive bladder

BACKGROUND: The Patient Perception of Intensity of Urgency Scale (PPIUS) is a patient-reported outcome instrument intended to measure the intensity of urgency associated with each urinary or incontinence episode. The objectives of this study were to assess the content validity, test-retest reliability, and acclimation effect of the PPIUS in overactive bladder (OAB) patients. METHODS: Patients undergoing treatment for OAB were recruited to participate in a non-interventional study by completing a three-day micturition diary including the PPIUS for three consecutive weeks. Following completion of the three-week study, participants from two select sites also completed a cognitive interview to assess their comprehension of the PPIUS. RESULTS: Thirty-nine participants successfully completed the three-week test-retest study; twelve of these participants completed the cognitive interview. Test-retest reliability was high based on intra-class correlation coefficient of 0.95. Among stable patients, the difference between the mean ratings of any two weeks was non-significant. Among the twelve interview participants, nine found it simple to choose a PPIUS rating for each of their micturition episodes and most found the urgency rating definitions consistent with their urgency experiences. CONCLUSIONS: The results demonstrated content validity based on qualitative interviews, and excellent test-retest reliability among stable patients. In addition, no acclimation effect was observed among stable patients. These findings support the use of the PPIUS as a reliable measure of urgency in both clinical trial and real life settings. The validity of PPIUS could be further established with future studies investigating the relationship between discretely graded urgency and incontinence continuum