Development of novel recombinant peroxidase secretion system from Pseudomonas putida for lignin valorisation.

Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin-depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.

Exploration of Tetrahydroisoquinoline- and Benzo[c]azepine-Based Sphingosine 1-Phosphate Receptor 1 Agonists for the Treatment of Multiple Sclerosis.

Because of the wide use of Fingolimod for the treatment of multiple sclerosis (MS) and its cardiovascular side effects such as bradycardia, second-generation sphingosine 1-phosphate receptor 1 (S1P1) agonist drugs for MS have been developed and approved by FDA. The issue of bradycardia is still present with the new drugs, however, which necessitates further exploration of S1P1 agonists with improved safety profiles for next-generation MS drugs. Herein, we report a tetrahydroisoquinoline or a benzo[c]azepine core-based S1P1 agonists such as 32 and 60 after systematic examination of hydrophilic groups and cores. We investigated the binding modes of our representative compounds and their molecular interactions with S1P1 employing recent S1P1 cryo-EM structures. Also, favorable ADME properties of our compounds were shown. Furthermore, in vivo efficacy of our compounds was clearly demonstrated with PLC and EAE studies. Also, the preliminary in vitro cardiovascular safety of our compound was verified with human iPSC-derived cardiomyocytes.

Influence of mobile phase composition on the analytical sensitivity of LC-ESI-MS/MS for the concurrent analysis of bisphenols, parabens, chlorophenols, benzophenones, and alkylphenols.

Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a "dilute and shoot" approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Analysis of Correlation Between Contrast and Component of Polylatic Acid Composite for Fused Deposition Modeling 3D Printing.

Additive manufacturing or three-dimensional (3D) printing is considered a disruptive technology for producing components with topologically optimized complex geometries as well as functionalities that are not achievable by traditional methods. 3D printing is expected to revolutionize the manufacturing of components. While several 3D printing systems are available, printing based on fused-deposition modeling (FDM) using thermoplastics is particularly widespread because of the simplicity and potential applicability of the method. In this study, we report the analysis of correlation between contrast and component of polylactic acid (PLA) based composite for FDM 3D printing. The pre-fabricated white composite and black composite were mixed in the fraction of 100:0, 90:10, 75:25, 50:50, 25:75, and 0:100% (v/v) and the obtained mixture was extruded using HX-35 3D filament extrusion line. The samples in different contrast were printed in disk like shape, and the gray scale filaments and 3D printed samples were measured the morphology and components using a field emission scanning electron microscope and energy dispersive X-ray spectroscopy. The CIE-lab values of the samples were measured using a colorimeter and the correlation between CIE-lab values and the components were analyzed. Although the component of Ti was linearly increased, the CIE-lab values show a clear exponential increase by increasing the white composite.

Liver Cirrhosis Patients Who Had Normal Liver Function Before Liver Cirrhosis Development Have the Altered Metabolic Profiles Before the Disease Occurrence Compared to Healthy Controls.

Liver cirrhosis (LC) is the final usual outcome of liver damage induced by various chronic liver diseases. Because of asymptomatic nature of LC, it is usually diagnosed at late and advanced stages, and patients are easy to miss the best timing for treatment. Thus, the early detection of LC is needed. In the prospective Korean Cancer Prevention Study-II (K-II), we aimed to identify valuable biomarkers for LC using metabolomics to distinguish subjects with incident LC (LC group) from subjects free from LC (control group) during a mean 7-year follow-up period. Metabolic alterations were investigated using baseline serum specimens acquired from 94 subjects with incident LC and 180 age- and sex-matched LC-free subjects via ultra-performance liquid chromatography (UPLC)-linear-trap quardrupole (LTQ)-Orbitrap mass spectrometry (MS). As a result of the metabolic analysis, 46 metabolites were identified. Among them, 11 and 18 metabolite level showed a significant increase and decrease, respectively, in the LC group compared to the control group. Nine metabolic pathways, including glyoxylate and dicarboxylate metabolism, amino acid metabolism, fatty acid metabolism, linoleic acid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism, were significantly different between the two groups. Logistic regression demonstrated that the LC emergence was independently affected by serum levels of myristic acid, palmitic acid, linoleic acid, eicosapentaenoic acid (EPA), lysophosphatidic acid (LPA) (18:1), glycolic acid, lysophosphatidylcholine (lysoPC) (22:6), and succinylacetone (R 2 = 0.837, P < 0.001). This prospective study revealed that dysregulation of various metabolism had the clinical relevance on the LC development. Moreover, myristic acid, palmitic acid, linoleic acid, EPA, LPA (18:1), glycolic acid, lysoPC (22:6), and succinylacetone were emerged as independent variables influencing the incidence of LC. The results support that the early biomarkers found in this study may useful for predicting and remedying the risk of LC.

Bacterial Valorization of Lignin: Strains, Enzymes, Conversion Pathways, Biosensors, and Perspectives.

Lignin, an aromatic polymer found in plants, has been studied for years in many biological fields. Initially, when biofuel was produced from lignocellulosic biomass, lignin was regarded as waste generated by the biorefinery and had to be removed, because of its inhibitory effects on fermentative bacteria. Although it has since proven to be a natural resource for bio-products with considerable potential, its utilization is confined by its complex structure. Hence, the microbial degradation of lignin has attracted researchers' interest to overcome this problem. From this perspective, the studies have primarily focused on fungal systems, such as extracellular peroxidase and laccase from white- and brown-rot fungi. However, recent reports have suggested that bacteria play an increasing role in breaking down lignin. This paper, therefore, reviews the role of bacteria in lignin and lignin-related research. Several reports on bacterial species in soil that can degrade lignin and their enzymes are included. In addition, a cellulolytic anaerobic bacterium capable of solubilizing lignin and carbohydrate simultaneously has recently been identified, even though the enzyme involved has not been discovered yet. The assimilation of lignin-derived small molecules and their conversion to renewable chemicals by bacteria, such as muconic acid and polyhydroxyalkanoates, including genetic modification to enhance their capability was discussed. This review also covers the indirect use of bacteria for lignin degradation, which is concerned with whole-cell biosensors designed to detect the aromatic chemicals released from lignin transformation.

Detection of Amyloid ß Using a Poly-L-Lysine Mediated Nanobiosensor.

Amyloid ß (Aß) peptide is secreted from the outside of neural cell by a neural signal pathway and it accumulated each other results in the highly toxicity amyloid plaque which is a critical causative factor in the pathogenesis of Alzheimer's Disease (AD). The peptide is considered to be a potential biomarker to diagnose AD. Here we introduce a novel poly-L-lysine (PLL) mediated nanobiosensor to detect Aß in vitro. The PLL molecules were utilized as a signal amplifier of Aß detection. The indirect enzyme-linked immunosorbent assay (ELISA) method and the sandwich ELISA method have tried to the detection of Aß. A commercially available ELISA plate was modified by PLL using a chemical agent and the amplified amino groups were activated by a functional group for the binding of Aß. The bound Aß was further modified with a primary antibody and fluorescence molecules conjugated secondary antibody by the traditional immunochemistry. In the result, the fluorescence intensity was increased by the increasing concentration of Aß, and the best Aß detection results were obtained in the PLL mediated indirect ELISA nanobiosensor. We expected that the present method would be optimized and applied for the detection of Aß in human fluid.

Development of Chemically Signal Amplified Nano-Biosensor Mediated by Poly-L-Lysine.

Amyloid ß (Aß) is considered to be one of a potential biomarker to monitor Alzheimer's Disease (AD) not only for diagnostic purposes but for early detection. Here we describe a novel nano-biosensor for Aß mediated by poly-L-lysine (PLL) which was used for the amplification of detection signal for Aß. The indirect enzyme-linked immunosorbent assay (ELISA) method was modified using PLL for the amplification of the Aß detection signal. A commercially available ELISA plate was modified by PLL using chemical agent and the amplified amino groups were activated by a chemical agent for the detection of Aß. The detection was carried out by the traditional immunochemistry using primary antibody and fluorescence molecules conjugated secondary antibody. In the result, the fluorescence intensity was increased by the increasing treated Aß amount, and the sensitivity was approximately 2 times higher in the concentration of 2 ng/mL Aß treatment, and approximately 4 times higher in the concentration of 200 ng/mL Aß treatment compare with that of indirect ELISA detection method. We suggest our novel signal amplification method for the Aß early detection.

Detection of Actin Filament and Cofilin Interaction Change by Actin Filament Curvature Decreasing.

Actin filament senses mechanical forces and it is transduced into biochemical signals during many cellular processes. In the disassembling process of actin filaments, cofilin plays a central role as the actin filament depolymerization. In this study, we evaluated a quantitative analysis of the actin filament-cofilin interaction change dependent upon the actin filament curvature decrease using atomic force microscopy (AFM) and a fabricated wave-like substrate. A wave-like substrate was fabricated by a maskless photo-lithography of a spin coated film on a glass substrate, and graphene oxide sheet was used for the decreasing of non-specific interaction between protein and the substrate. By single-molecule force spectroscopy, we determined rupture force of actin filament-cofilin binding on the wave-like substrate and a flat substrate. The rupture force of actin filament-cofilin binding at the curvature of -1.35 µm-1 showed a value approximately 4 times higher than the rupture force at the curvature of -0.15 µm-1. The present study will provide the possibility and quantitative evidence that mechanical stress on cytoskeletal filaments can modulate how they interact with their binding proteins.

Probing Amyloid ß and the Antibody Interaction Using Atomic Force Microscopy.

Amyloid ß (Aß) peptide is considered to be the critical causative factor in the pathogenesis of Alzheimer's disease (AD) because the hydrophilic molecules accumulated outside of the neural cells and results in the formation of highly toxicity amyloid plaque. In this study, we probed the interaction between Aß and the antibody using atomic force microscopy (AFM). We compared two kinds of antibodies which are the antibody for Aß 1-42 (antibody42) and the antibody for Aß 1-16 (antibody16). To detect the interaction between Aß and the antibodies, the single molecular force spectroscopy was carried out using Aß modified glass substrate and the antibodies modified AFM probes. In the results, the single Aß-antibody42 dissociation constant was estimated to be 5.2 × 10-3 s-1 and the single Aß-antibody16 dissociation constant was 2.8×10-2 s-1. The Aß-antibody42 showed 5.3 times longer bond life time compare with Aß-antibody16. It suggested that antibody42 is better choice for the Aß sensor development.

Analysis of metabolites and metabolic pathways in breast cancer in a Korean prospective cohort: the Korean Cancer Prevention Study-II.

INTRODUCTION: Since blood is in contact with all tissues in the body and is considered to dynamically reflect the body's pathophysiological status, serum metabolomics changes are important and have diagnostic value in early cancer detection. OBJECTIVES: In this prospective study, we investigated the application of metabolomics to differentiate subjects with incident breast cancer (BC) from subjects who remained free of cancer during a mean follow-up period of 7 years with the aim of identifying valuable biomarkers for BC. METHODS: Baseline serum samples from 84 female subjects with incident BC (BC group) and 88 cancer-free female subjects (control group) were used. Metabolic alterations associated with BC were investigated via metabolomics analysis of the baseline serum samples using ultra-performance liquid chromatography-linear-trap quadrupole-Orbitrap mass spectrometry. RESULTS: A total of 57 metabolites were identified through the metabolic analysis. Among them, 20 metabolite levels were significantly higher and 22 metabolite levels were significantly lower in the BC group than in the control group at baseline. Ten metabolic pathways, including amino acid metabolism, arachidonic acid (AA) metabolism, fatty acid metabolism, linoleic acid metabolism, and retinol metabolism, showed significant differences between the BC group and the control group. Logistic regression revealed that the incidence of BC was affected by leucine, AA, prostaglandin (PG)J2, PGE2, and γ-linolenic acid (GLA). CONCLUSIONS: This prospective study showed the clinical relevance of dysregulation of various metabolisms on the incidence of BC. Additionally, leucine, AA, PGJ2, PGE2, and GLA were identified as independent variables affecting the incidence of BC.