Systematic Engineering of Genistein Biosynthetic Pathway through Genetic Regulators and Combinatorial Enzyme Screening.

Microbial production of genistein, an isoflavonoid primarily found in soybeans, is gaining prominence in the food industry due to its significant nutritional and health benefits. However, challenges arise in redesigning strains due to intricate regulatory nodes between cell growth and genistein production and in systematically exploring core enzymes involving genistein biosynthesis. To address this, this study devised a strategy that simultaneously and precisely rewires flux at both acetyl-CoA and malonyl-CoA nodes toward genistein synthesis. In particular, naringenin, the primary precursor of genistein, was accumulated 2.6 times more than the unoptimized strain through transcriptional repressor-based genetic regulators. Building upon this, a combination of isoflavone synthase and cytochrome P450 reductase with the remarkable conversion of naringenin to genistein was screened from enzyme homologue libraries. The integrated metabolic engineering strategy yields the highest reported production (98 mg/L of genistein) to date, providing a framework for the biosynthesis of diverse flavonoids, including genistein.

[Experiences of Transitional Care for Medicaid Case Managers].

PURPOSE: This phenomenological study tried to understand the essence of the transitional care experience of medicaid case managers and its structural meaning. In addition, it was attempted to establish a system of transitional care and seek support measures for medicaid case managers. METHODS: The participants of this study were 7 medicaid case managers who had spent more than 1 year and 6 months in medicaid pilot project. Data were collected with individual in-depth interviews from June to December 2021. The data were analyzed by Giorgi's phenomenological analysis method. RESULTS: The seven constituents derived from the results of this study were 'struggle to establish a living environment', 'dedication to supporting independent living', 'anxiety about safety', 'pressure on care responsibilities', 'distress in building the pilot project', 'pride in role', and 'expectation for improvement'. CONCLUSION: The study results provide a comprehensive understanding of the transition care reality for medicaid case managers. They also shed light on managers' perceptions and attitudes. These findings can serve as fundamental information for establishing support measures for medicaid case managers and transitional care systems.

Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches.

BACKGROUND: Synthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive sensor modules for use in synthetic biology applications. However, tuning the fold-change of riboswitch circuits is challenging because a deep understanding of the riboswitch mechanism and screening of mutant libraries is generally required. Therefore, novel molecular parts and strategies for straightforward tuning of the fold-change of riboswitch circuits are needed. RESULTS: In this study, we devised a toehold switch-based modulator approach that combines a hybrid input construct consisting of a riboswitch and transcriptional repressor and de-novo-designed riboregulators named toehold switches. First, the introduction of a pair of toehold switches and triggers as a downstream signal-processing module to the hybrid input for coenzyme B12 resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on balancing the expression levels of the RNA components greatly improved the fold-change from 260- to 887-fold depending on the promoter and host strain. Further characterizations confirmed low leakiness and high orthogonality of five toehold switch pairs, indicating the broad applicability of this strategy to riboswitch tuning. CONCLUSIONS: The toehold switch-based modulator substantially improved the fold-change compared to the previous sensors with only the hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.

Roles of 14-3-3ß and γ in regulation of the glucocorticoid receptor transcriptional activation and hepatic gluconeogenesis.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that mediates the effects of glucocorticoids, and plays a crucial role in cell growth, development, inflammation, and gluconeogenesis. The 14-3-3 proteins bind to target proteins via phosphorylation, and influence many cellular events by altering their subcellular localization or by acting as chaperones. However, the mechanisms by which 14-3-3 proteins regulate GR transactivation and their involvement in gluconeogenesis remain uncharacterized. We found that 14-3-3ß and γ increased GR transcriptional activity and the promoter activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase in the presence of glucocorticoids. Inhibition of the endogenous 14-3-3ß and γ decreased dexamethasone- and cAMP-stimulated PEPCK expression. Further, both 14-3-3ß and γ increased glucose production in response to glucocorticoids. Our findings suggest that 14-3-3ß and γ function as positive regulators of GR transactivation and glucocorticoid-mediated hepatic gluconeogenesis.

Enzymatic susceptibility of wheat gluten after subcritical water treatment.

Subcritical water (SCW) hydrolysis is an alternative to traditional methods of protein hydrolysis that uses water as a reaction medium. In this study, the effect of SCW treatment on heat-induced conformational changes in wheat gluten and its relation to enzymatic susceptibility were investigated. The degree of deamidation increased rapidly from 12.5 to 47.4% with increase in the temperature range of 160-220 °C. Protein solubility increased in a similar pattern with degree of deamidation and almost all protein was solubilized after treatment with SCW at 200 °C. SCW treatment in a particular time-temperature combination results in a significant decrease in enzymatic susceptibility. After SCW treatment at 220 °C for 20 min, enzymatic susceptibility of gluten protein was exceedingly decreased to nearly complete loss. Because of excess degradation and deamidation and small molecular size (less than 6500 Da) many hydrolysis sites disappear and are difficult to access by protease.

Photoelectrochemical polymerization of thiophene on self-assembled RuL2(NCS)2/di(3-aminopropyl)viologen on indium thin oxide.

Polythiophene layers were formed on self-assembled monolayers (SAMs)/indium tin oxide (ITO) using photoelectrochemical polymerization. The SAMs on ITO was prepared using Ru(4,4'-dicarboxylic acid-2,2'-bipyridine)2(NCS)2 and di(3-aminopropyl)viologen. The photoelectrochemically polymerized polythiophene layers on SAMs/ITO were characterized using UV-vis. absorption spectroscopy, atomic force microscopy, scanning electron microscopy, and cyclic voltammetry. The polymer layers have thickness of 360 nm, a dense surface morphology, optical gap of 2.38 eV, highest occupied molecular orbital of -5.2 eV and lowest unoccupied molecular orbital of -2.82 eV. In photoelectrochemical cells, the polythiophene on SAMs/ITO electrode showed a photocurrent of 5 microA/cm2.

Enhanced photocurrent in the RuL2(NCS)2/ di-(3-aminopropyl)-viologen/Au nanoparticles/ITO system: use of Au nanoparticles for retardation of the back electron transfer reaction.

This paper reports the use of Au nanoparticles (NPs) as electron transfer bridge layers to improve the photocurrent of viologen/Ru complex-based photoelectrochemical cells. The Ru complex/ viologen/Au NPs on electrodes were prepared using self-assembled monolayers. The cell system showed an excellent photocurrent of 25 nA/cm2 under the 1.5 air mass condition (I = 100 mW/cm2), which is five times greater than Au NPs due to the reduced recombination reaction.

Conjugated linoleic acid reduces adipose depots without reduction of stearoyl coenzyme A desaturase 1 gene expression.

AIMS AND METHODS: This study investigated the effects of conjugated linoleic acid (CLA) on the body weight, fat deposition and the expression of stearoyl coenzyme A desaturase 1 (SCD1) in the livers of male ICR mice that were fed with either beef tallow (BT) or fish oil (FO) supplemented with CLA. Mice weighing 25-30 g were divided into four groups, BT and BT supplemented with 0.5% CLA (BTC), FO and FO supplemented with 0.5% CLA (FOC). Each group consisted of 8 mice, and they were fed the experimental diets for 4 weeks. The experimental diets were composed of 59.18% carbohydrates, 19.73% proteins and 21.09% fat in terms of their contributions to total calories, and other nutrients were identical. CLA was added to the diets of the CLA supplementation groups at 0.5% (w/w). The measurement of triglyceride (TG) was done by using a kit. Fatty acid compositions were analyzed in both the plasma and the liver using a gas chromatograph. The levels of SCD1 expression were analyzed by RT-PCR in the liver. RESULTS: No significant difference was found in the levels of food intake, body weight and food efficiency among experimental groups. However, the levels of both epididymal and visceral fat weight were significantly lower in the CLA-supplemented groups, BTC and FOC (p < 0.05). The level of the triglyceride concentration was also significantly lower in the CLA-supplemented groups (p < 0.05). The levels of the liver SCD1 gene expression were also higher in the CLA-supplemented groups. However, significant inhibition of conversion from C16:0 to C16:1 was found in both fatty acid compositions of the liver (p < 0.05). CONCLUSION: Addition of 0.5% CLA did not inhibit the liver SCD1 gene expression; however, it showed decreasing effects on the fat pad weight, the concentration of TG and on fatty acid composition, leading to a decrease in the fat depositions. Such effects were clearer when CLA was supplemented to BT rather than FO.