CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.

Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.

Synthetic biology tools for engineering Corynebacterium glutamicum.

Corynebacterium glutamicum is a promising organism for the industrial production of amino acids, fuels, and various value-added chemicals. From the whole genome sequence release, C. glutamicum has been valuable in the field of industrial microbiology and biotechnology. Continuous discovery of genetic manipulations and regulation mechanisms has developed C. glutamicum as a synthetic biology platform chassis. This review summarized diverse genomic manipulation technologies and gene expression tools for static, dynamic, and multiplex control at transcription and translation levels. Moreover, we discussed the current challenges and applicable tools to C. glutamicum for future advancements.

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

Effect of the Strategic Thinking, Problem Solving Skills, and Grit on the Disaster Triage Ability of Emergency Room Nurses.

In this descriptive study, we aimed to identify factors related to emergency room nurses' disaster triage ability. A total of 166 nurses who worked for emergency departments of general hospitals completed a structured questionnaire consisting of the Disaster Triage Ability Scale (DTAS), the Strategic Thinking Scale (STS), the Problem-Solving Inventory (PSI), and the Original Grit Scale (Grit-O). The data were analyzed using SPSS/WIN 25.0 by means of descriptive statistics, t-test, one-way ANOVA, the Scheffé post hoc test, Pearson's correlation coefficients, and stepwise multiple regression. Participants' DTAS averaged 14.03 ± 4.28 (Range 0-20) and showed a statistically significant difference according to their experience of triage education (t = 2.26, p = 0.022) as a disaster triage-related attribute. There were significant correlations among DTAS and confidence in the PSI (r = 0.30, p < 0.001), the approach-avoidance style in the PSI (r = -0.28, p < 0.001), and futurism in the STS (r = 0.19, p = 0.019). The strongest predictor was confidence in the PSI; in addition, 14.1% of the DTAS was explained by confidence in the PSI, approach-avoidance in the PSI, and futurism in the STS. Emergency room nurses who received triage education showed a higher level of the DTAS and their DTAS could be explained by problem-solving skills and strategic thinking. Therefore, it is necessary to develop and implement triage education programs integrated with stress management to improve the approach-avoidance style to ensure better problem-solving skills and to utilize various training methods to enhance confidence to improve problem-solving skills and futurism as part of strategic thinking.

Synthetic cellular communication-based screening for strains with improved 3-hydroxypropionic acid secretion.

Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.

Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

Determination of inflammation-related lipids in depressive rats by on-line supercritical fluid extraction-supercritical fluid chromatography-tandem mass spectrometry.

An on-line supercritical fluid extraction coupled with supercritical fluid chromatography-quadrupole tandem mass spectrometry method was developed to determine lipids related to inflammation in brain tissues of depressed rats. The analysis of 23 lipids from extraction to separation and detection only took 15 min and required 1 mg of brain tissue powder. The matrix effect of the on-line method for endogenous lipids was systematically investigated, and targeted lipids were quantified by matrix effect corrected calibration curves in this study. The on-line method was comprehensively optimized and evaluated. All calibration curves for lipids showed good linearity (correlation coefficient >0.99). The limits of detection and the limits of quantification were in the range of 0.0261-0.396 pg and 0.0791-1.20 pg. The recoveries and the matrix effect were in the range of 85.3-117.5% and 51.9-176.6%, respectively. The relative standard deviations of precision ranged from 2.7 to 14.2%, with accuracies higher than 87.2%. Compared with liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry method, the on-line method obtained higher recovery and sensitivity with significantly reduced analytical time, manual operations, and sample amounts. Finally, this on-line method was applied to analyses of brain tissues of depressed rats. Six pro-inflammatory lipids increased in depressed rats, while six anti-inflammatory lipids decreased. Liquiritin and fluoxetine were presumed to promote a similar synthesis of anti-inflammatory lipids. Based on the results, this on-line method showed great promise in analyzing lipids in complex biological samples.

A high coverage pseudotargeted lipidomics method based on three-phase liquid extraction and segment data-dependent acquisition using UHPLC-MS/MS with application to a study of depression rats.

Pseudotargeted analysis combines the advantages of untargeted and targeted lipidomics methods based on chromatography-mass spectrometry (MS). This study proposed a comprehensive pseudotargeted lipidomics method based on three-phase liquid extraction (3PLE) and segment data-dependent acquisition (SDDA). We used a 3PLE method to extract the lipids with extensive coverage from biological matrixes. 3PLE was composed of one aqueous and two organic phases. The upper and middle organic phases enriched neutral lipids and glycerophospholipids, respectively, combined and detected together. Besides, the SDDA strategy improved the detection of co-elution ions in the lipidomics analysis. A total of 554 potential lipids were detected by the developed approach in both positive and negative modes using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Compared with the conventional liquid-liquid extraction (LLE) approaches, including methyl tert-butyl ether (MTBE) and Bligh-Dyer (BD) methods, 3PLE combined with SDDA significantly increased the lipid coverage 87.2% and 89.7%, respectively. Also, the proposed pseudotargeted lipidomics approach exhibited higher sensitivity and better repeatability than the untargeted approach. Finally, we applied the established pseudotargeted method to the plasma lipid profiling from the depressed rats and screened 61 differential variables. The results demonstrated that the pseudotargeted method based on 3PLE and SDDA broadened lipid coverage and improved the detection of co-elution ions with excellent sensitivity and precision, indicating significant potential for the lipidomics analysis.

Engineering Vibrio sp. SP1 for the production of carotenoids directly from brown macroalgae.

Macroalgae is regarded as a promising third-generation marine biomass that can be utilized as a sustainable feedstock for bio-industry due to the high sugar level and absence of lignin. Alginate, composed of 1,4-linked D-mannuronate (M) and L-guluronate (G), is one of the major carbohydrates in brown macroalgae. It is difficult to be assimilated by most industrial microorganisms. Therefore, developing engineered microorganisms that can utilize alginate as a feedstock in order to produce natural products from marine biomass is critical. In this study, we isolated, characterized, and sequenced Vibrio sp. SP1 which rapidly grows using alginate as a sole carbon source. We further engineered this strain by introducing genes encoding enzymes under the control of synthetic expression cassettes to produce lycopene and ß-carotene which are attractive phytochemicals owing to the antioxidant property. We confirmed that the engineered Vibrio sp. SP1 could successfully produce 2.13 ± 0.37 mg L-1 of lycopene, 2.98 ± 0.43 mg L-1 of ß -carotene, respectively, from 10 g L-1 of alginate as a sole carbon source. Furthermore, our engineered strain could directly convert 60 g L-1 of brown macroalgae Sargassum fusiforme into 1.23 ± 0.21 mg L-1 of lycopene without any pretreatment which had been vitally required for the previous productions. As the first demonstrated strain to produce high-value product from Sargassum, Vibrio sp. SP1 is evaluated to be a desirable platform for the brown macroalgae-based biorefinery.

Synthetic protein quality control to enhance full-length translation in bacteria.

Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated messenger RNAs, inevitably generating nonfunctional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA-RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6- to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant protein production but also efficiencies of metabolic pathways.

Clinical Outcomes of Enteral Feeding Protocol Via Percutaneous Endoscopic Gastrostomy: A Single-Center, Retrospective Study.

BACKGROUND: The development of the endoscopic technique has resulted in an increasing number of patients undergoing percutaneous endoscopic gastrostomy (PEG) insertion; however, the protocols for increasing the volume of feeding formula after PEG insertion have not been established. Therefore, we compared the clinical outcomes of patients receiving low- and high-volume increase in enteral feeding formula. METHODS: A total of 215 patients who underwent PEG insertion between January 2016 and March 2019 were included. They were divided into 2 groups according to the increase in volume of feeding formula: the low-volume group (n = 135) received ≤150 mL/d, and the high-volume group (n = 80) received ≥300 mL/d. Patient characteristics, procedure, and feeding-related clinical outcomes were retrospectively reviewed using medical records. RESULTS: The adverse events of the feeding protocol did not significantly differ between the 2 groups. The number of days needed to attain the calorie targets was significantly lower in the high-volume group than in the low-volume group (5.4 ± 3.0 vs 2.4 ± 1.5; P < .001). The duration of supplemental parenteral nutrition and the length of hospitalization were also significantly lower in the high-volume group (3.9 ± 3.3 vs 1.2 ± 2.2; P < .001 and 5.8 ± 2.7 vs 4.6 ± 2.6; P = .007, respectively). CONCLUSION: To rapidly attain the calorie targets in appropriately selected patients with PEG insertion, a high-volume increase in daily feeding can safely be recommended given the favorable outcomes.

A green and efficient pseudotargeted lipidomics method for the study of depression based on ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

The pseudotargeted lipidomics method integrates the advantages of untargeted andtargeted lipidomics methods as a novel emerging approach. In this study, a green andefficient pseudotargeted lipidomics method based on ultra-high performancesupercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) wasdeveloped. The tandem mass spectra of the analytes were obtained by using UHPSFCwith quadrupole-time of flight MS (Q-TOF MS) in MS E mode and the multiplereaction monitoring (MRM) transitions of the lipidome were defined. Then, thecandidate MRM transitions were verified by UHPSFC with triple quadrupole massspectrometry (QqQ MS) in the scheduled MRM mode. In total, 758 potential lipidscorresponding to 509 and 249 MRM transitions were detected within 8 min in positiveand negative modes, respectively. The established pseudotargeted lipidomics methodwas validated to have excellent analytical characteristics. Compared with thepseudotargeted method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), the UHPSFC-MS/MS-basedpseudotargeted method not only reduced the analytical time by half but also improvedthe sensitivity and resolution for most analytes, especially had better separation forlipid isomers. Besides, the UHPSFC-MS/MS-based pseudotargeted method showedhigher sensitivity and better repeatability for most analytes than the UHPSFC-MS/MS-based untargeted method. The established method was finally applied to investigatingthe lipid profiles of the plasma from the depressed rats and 33 differential variableswere screened, which related to three metabolic pathways. The results indicated thatthe UHPSFC-MS/MS-based pseudotargeted method is reliable and efficient and couldbe used in the lipidomics studies.

Enhanced pseudotargeted analysis using a segment data dependent acquisition strategy by liquid chromatography-tandem mass spectrometry for a metabolomics study of liquiritin in the treatment of depression.

An enhanced pseudotargeted method using a segment data-dependent acquisition mode based on ultra-high performance liquid chromatography-tandem mass spectrometry was developed. This segment data dependent acquisition-based pseudotargeted method could improve the detection of co-eluted ions and extend the coverage of analytes. A set of 502 multiple reaction monitoring channels were obtained by this segment strategy, which was twice the number created by the traditional data-dependent acquisition mode. Compared with the untargeted method, the pseudotargeted profiling demonstrated higher sensitivity and higher precision. More than 90% of the metabolites detected by the enhanced pseudotargeted method had relative standard deviations less than 15%. The segment data dependent acquisition-based pseudotargeted method was successfully applied to the metabolomics study of the depressed rats with the treatment of liquiritin. Forty-seven differential metabolites were screened and five metabolic pathways were found to be related to depression including retinol metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, terpenoid backbone biosynthesis, and lysine degradation. The segment data dependent acquisition-based pseudotargeted method widened the coverage of metabolites with good sensitivity and precision, which exhibited great potential in the discovery of differential metabolites in metabolomics studies.

[Recent advances in sample preparation techniques for the lipid profiling based on chromatography-mass spectrometry].

As the main structural components of cellular and sub-cellular membranes, lipids are a major source of energy and play an important role in various biological processes such as cellular signaling. Lipid profiling has attracted increasing attention in recent years, and chromatography-mass spectrometry techniques for lipid profiling occupy a dominant position in this regard. Sample preparation, which aims at enriching trace substances and reducing matrix interferences, is a crucial step in the analysis of lipids for the intricacy of sample matrices. Here, we review recent developments in sample preparation techniques based on chromatography-mass spectrometry and their application to lipid profiling. Various sample preparation techniques are described and summarized. The extraction methods based on liquid phase included liquid-liquid extraction and single organic solvent extraction. The extraction methods based on solid phase involved solid-phase extraction and solid-phase microextraction. The field-assisted extraction methods cover supercritical fluid extraction, pressurized liquid extraction, microwave-assisted extraction, and ultrasound-assisted extraction. Moreover, online coupling sample preparation methods and sample preparation methods for in vivo lipid analysis are presented. Finally, the problems and trends in sample preparation techniques for lipid profiling based on chromatography-mass spectrometry are discussed. It is believed that efficient development of sample preparation techniques would help improve the sensitivity and selectivity of lipid profiling as well as the analysis speed.

A novel formulation significantly increases the cytotoxicity of flaxseed orbitides (linusorbs) LOB3 and LOB2 towards human breast cancer MDA-MB-231 cells.

Flaxseed orbitides (linusorbs) are a family of N to C linked bioactive cyclic octa-, nona-, and decapeptides present in flaxseed oil. They are highly hydrophobic and thermally stable. Our previous studies showed that [1-9-NαC]-linusorb B3 (LOB3) and [1-9-NαC]-linusorb B2 (LOB2) exhibited cytotoxic effects towards human breast cancer HER2-subtype Sk-Br-3 cells at a concentration of ~400 µM. However, this high concentration significantly limits their potential clinical applications. In the current study, we developed a novel polyethylene glycol-based formulation for linusorbs and showed that both LOB3 and LOB2, especially LOB3, exhibited strong cytotoxicity towards human breast cancer triple-negative-subtype MDA-MB-231 cells at low nanomolar concentrations.

Synthetic biology for evolutionary engineering: from perturbation of genotype to acquisition of desired phenotype.

With the increased attention on bio-based industry, demands for techniques that enable fast and effective strain improvement have been dramatically increased. Evolutionary engineering, which is less dependent on biological information, has been applied to strain improvement. Currently, synthetic biology has made great innovations in evolutionary engineering, particularly in the development of synthetic tools for phenotypic perturbation. Furthermore, discovering biological parts with regulatory roles and devising novel genetic circuits have promoted high-throughput screening and selection. In this review, we first briefly explain basics of synthetic biology tools for mutagenesis and screening of improved variants, and then describe how these strategies have been improved and applied to phenotypic engineering. Evolutionary engineering using advanced synthetic biology tools will enable further innovation in phenotypic engineering through the development of novel genetic parts and assembly into well-designed logic circuits that perform complex tasks.

Advances of supercritical fluid chromatography in lipid profiling.

Supercritical fluid chromatography (SFC) meets with great favor due to its high efficiency, low organic solvent consumption, and the specialty for the identification of the isomeric species. This review describes the advances of SFC in targeted and untargeted lipid profiling. The advancement of the SFC instruments and the stationary phases are summarized. Typical applications of SFC to the targeted and untargeted lipid profiling are discussed in detail. Moreover, the perspectives of SFC in the lipid profiling are also proposed. As a useful and promising tool for investigating lipids in vitro and in vivo, SFC will predictably obtain further development.

The investigation of protective effects of isosteviol sodium on cerebral ischemia by metabolomics approach using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Cerebral ischemia remains a major cause of mortality and a long-term disability with limited therapies. Isosteviol sodium (STV-Na) was proved to exert significant protective effects on cerebral ischemia, but the protective mechanism was not understood. In this study, the protective effects of STV-Na on cerebral ischemia were investigated by the metabolomics approach based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry technique. The models of ischemic rats were established and the brain tissues were employed for metabolomics analyses. The principal component analysis showed that the model group was clearly separated from the sham group, while both STV-Na and edaravone groups were located between the sham and the model groups, which indicated that STV-Na as well as edaravone had protective effects on cerebral ischemia. Eighteen differential metabolites which had significant differences between the sham and the model groups were screened and identified. After the administration of STV-Na, all 18 differential metabolites were regulated to the levels between the sham and the model groups, and 12 of them presented significant differences between the model and STV-Na groups. The pathway analysis indicated that the protective effects of STV-Na on cerebral ischemia might be associated with the regulation of several metabolic pathways, i.e. glycerophospholipid metabolism, arachidonic acid metabolism and linoleic acid metabolism.

Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

Diffusiophoretic exclusion of colloidal particles for continuous water purification.

It has been observed that colloidal particles are anomalously repelled from the interface of nanoporous materials and water by up to hundreds of micrometers even if there is no additional external field present. Recently, the physical origin of this anomalous repulsion has turned out to be diffusiophoretic migration triggered by an ion exchange process through the interface. Since the repulsive force is induced by a salt gradient only, the phenomenon can be applied to a microscale water purification platform without the need for any external power sources. In this work, we suggest a micro/nanofluidic device for continuous water purification utilizing long-range diffusiophoretic migration around ion exchangeable surfaces. An ion concentration boundary layer was characterized by the Sherwood number (Sh) which is a key dimensionless number to describe the purification process. Depending on Sh, we have theoretically and experimentally demonstrated that long-range diffusiophoretic exclusion can be used for continuous water purification. Finally, our platform can be used as a highly energy-efficient and portable water treatment option for operations such as purification, disinfection, water softening, etc.