Loop-Mediated Isothermal Amplification and Lateral Flow Immunochromatography Technology for Rapid Diagnosis of Influenza A/B.

Influenza viruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Rapid detection of influenza viruses is essential for accurate diagnosis and the initiation of appropriate treatment. We developed a loop-mediated isothermal amplification and lateral flow assay (LAMP-LFA) capable of simultaneously detecting influenza A and influenza B. Primer sets for influenza A and influenza B were designed to target conserved regions of segment 7 and the nucleoprotein gene, respectively. Optimized through various primer set ratios, the assay operated at 62 °C for 30 min. For a total of 243 (85 influenza A positive, 58 influenza B positive and 100 negative) nasopharyngeal swab samples, the performance of the influenza A/B multiplex LAMP-LFA was compared with that of the commercial AllplexTM Respiratory Panel 1 assay (Seegene, Seoul, Korea). The influenza A/B multiplex LAMP-LFA demonstrated a specificity of 98% for the non-infected clinical samples, along with sensitivities of 94.1% for the influenza A clinical samples and 96.6% for the influenza B clinical samples, respectively. The influenza A/B multiplex LAMP-LFA showed high sensitivity and specificity, indicating that it is reliable for use in a low-resource environment.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

Production of 2'-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

The effects of a community-based walking program on walking ability and fall-related self-efficacy of chronic stroke patients.

The objectives of this study were to evaluate the effects of community-based walking training (CWT) on the walking ability and fall-related self-efficacy of chronic stroke patients and compare the effects of CWT to the conventional walking programs in stroke patients. Previous studies focused on walking speed, walking endurance, and balance. However, no studies have examined the changes in fall-related self-efficacy after CWT. In order to achieve purpose of this study, 45 chronic stroke patients, who were hospitalized at National Rehabilitation Center, were randomly divided into the CWT group (CWTG, n=15), the treadmill walking training group (TWTG, n=15), and the control group (CG, n=15). The treatment was conducted 3 times per week (30 min each) for 4 weeks. CWT was carried out by gradually increasing the difficulty level in various environments outside the hospital room. The results revealed that the CWTG was more effective in enhancing the walking ability and fall-related self-efficacy than the TWTG and the CG. These findings demonstrated that the CWTG increased the walking ability and fall-related self-efficacy of chronic stroke patients. Therefore, we suggest that adding CWT to standard rehabilitation might be an effective method for improving walking ability and fall-related self-efficacy in chronic stroke patients.

Effects of surface roughness on hydrogen gas sensing properties of single Pd nanowires.

We report on the effects of surface roughness resulting from an ion milling technique on the hydrogen gas sensing performance of a single Pd nanowire grown by electrodeposition into nanochannels in anodized aluminum oxide templates. A combination of electron beam lithography and a lift-off process was utilized to fabricate four-terminal devices based on individual Pd nanowires. These results are the first demonstration of the effect of ion milling on the response time in a single Pd nanowire used as a hydrogen sensor. The response time of the single Pd nanowire surface-treated by ion milling was 20 times faster than that of a sample without surface treatment. The faster response time was due to the surface roughness effects of the surface treatment, an increase in the surface-to-volume ratio of the ion-milled nanowire.

Low-dimensional palladium nanostructures for fast and reliable hydrogen gas detection.

Palladium (Pd) has received attention as an ideal hydrogen sensor material due to its properties such as high sensitivity and selectivity to hydrogen gas, fast response, and operability at room temperature. Interestingly, various Pd nanostructures that have been realized by recent developments in nanotechnologies are known to show better performance than bulk Pd. This review highlights the characteristic properties, issues, and their possible solutions of hydrogen sensors based on the low-dimensional Pd nanostructures with more emphasis on Pd thin films and Pd nanowires. The finite size effects, relative strengths and weaknesses of the respective Pd nanostructures are discussed in terms of performance, manufacturability, and practical applicability.

Hydrogen gas sensing properties of PdO thin films with nano-sized cracks.

We report on a novel method for the fabrication of highly sensitive hydrogen gas sensors based on palladium oxide thin films and have investigated their hydrogen sensing properties and nanostructures. To our knowledge, this is the first report on the use of palladium oxide and reduced palladium thin films as hydrogen sensors. The palladium oxide thin films were deposited on thermally oxidized Si substrates using a reactive direct current (DC) magnetron sputtering system. Considerable changes in the resistance of the palladium oxide thin films were observed when they were initially exposed to hydrogen gas, as a result of the reduction process. After the initial exposure to hydrogen gas of PdO(30%), its sensitivity increased up to approximately 4.5 x 10(3)%. The morphology of the PdO surface was analyzed using a scanning electron microscope (SEM), in order to investigate the interactions between palladium oxide and hydrogen. The SEM images showed a large number of nano-sized cracks on the surface of the palladium oxide during the reduction process, which acted to increase the effective surface-to-volume ratio. The response behaviors of the reduced Pd films to hydrogen gas were reversible and had an enhanced sensing property when compared with those of the pure Pd films. In addition, their sensitivities and response times were improved due to the nano-sized cracks on the surfaces. The results demonstrate that palladium oxide and reduced palladium thin films can be applied for use in highly sensitive hydrogen sensors.

Individual Pd nanowire hydrogen sensors fabricated by electron-beam lithography.

We report on the hydrogen gas (H2) sensing performance of lithographically patterned Pd nanowires as a function of the nanowire thickness and H2 concentration. A combination of electron-beam lithography and a lift-off process has been utilized to fabricate four-terminal devices based on individual Pd nanowires with width w = 300 nm, length l = 10 microm, and thickness t = 20-400 nm from continuous Pd films. The variation of the resistance and sensitivity at 20 000 ppm H2 of Pd nanowires was found to be much lager than at 10 000 ppm H2, which can be explained by an alpha-beta phase transition occurring at 20 000 ppm H2. This is confirmed by the observation of hysteresis behavior in the resistance versus H2 concentration for Pd thin films. The response time was found to decrease with decreasing thickness regardless of H2 concentration due to a higher surface-to-volume ratio and a higher clamping effect. A single Pd nanowire with t = 100 nm was found to successfully detect H2 at a detection limit of 20 ppm. Our results suggest that lithographically patterned Pd nanowires can be used as hydrogen gas sensors to quantitatively detect H2 over a wide range of concentrations.

Finite size effect on hydrogen gas sensing performance in single Pd nanowires.

We present the hydrogen sensing performance of individual Pd nanowires grown by electrodeposition into nanochannels of anodized aluminum oxide (AAO) templates investigated as a function of the nanowire diameter. Four-terminal devices based on individual Pd nanowires were found to successfully detect hydrogen gas (H(2)). Our experimental results show that the H(2) sensing sensitivity increases and the response time decreases with decreasing diameter of Pd nanowires with d = 400, 200, 80 and 20 nm, due to the high surface-to-volume ratio and short diffusion paths, respectively. This is in qualitatively good agreement with simulated results obtained from a theoretical model based on a combination of the rate equation and diffusion equation.