Engineering of two thiamine diphosphate-dependent enzymes for the regioselective condensation of C1-formaldehyde into C4-erythrulose.

A number of carboligases, which catalyze condensation of C1- and/or C2-aldehydes into multi-carbon products, have been reported. However, their catalytic activities and/or regioselectivities remained rather low. Thereby, this study has focused on engineering of C1 and C2 carboligases for the regioselective condensation of C1-formaldehyde into C4-erythrulose via C2-glycolaldehyde. The crystal structure of the glyoxylate carboligase from Escherichia coli (EcGCL) was elucidated in complex with glycolaldehyde. A structure-guided rationale generated several mutants, one of whose catalytic activity reached 15.6 M-1·s-1, almost 10 times greater than the wild-type enzyme. Another variant (i.e., EcGCL_R484M/N283Q/L478M/M488L/R284K) has shown significantly increased stability to the glycolaldehyde toxicity, enabling production of glycolaldehyde to 31 mM from 75 mM formaldehyde (conversion: 83 %). Besides, the E1 subunit of α-ketoglutarate dehydrogenase complex from Vibrio vulnificus (VvSucA) was engineered as a regiospecific C2 carboligase for condensation of glycolaldehyde into erythrulose. The combination of EcGCL_R484M/N283Q/L478M/M488L/R284K and VvSucA_K228L led to the cascade production of erythrulose to 8 mM from 90 mM formaldehyde via glycolaldehyde without byproduct formation. This study will contribute to valorization of C1 gases into industrially relevant multi-carbon products in an environment-friendly way.

Suaeda glauca Attenuates Liver Fibrosis in Mice by Inhibiting TGFß1-Smad2/3 Signaling in Hepatic Stellate Cells.

Chronic liver injury due to various hepatotoxic stimuli commonly leads to fibrosis, which is a crucial factor contributing to liver disease-related mortality. Despite the potential benefits of Suaeda glauca (S. glauca) as a natural product, its biological and therapeutic effects are barely known. This study investigated the effects of S. glauca extract (SGE), obtained from a smart farming system utilizing LED lamps, on the activation of hepatic stellate cells (HSCs) and the development of liver fibrosis. C57BL/6 mice received oral administration of either vehicle or SGE (30 or 100 mg/kg) during CCl4 treatment for 6 weeks. The supplementation of SGE significantly reduced liver fibrosis induced by CCl4 in mice as evidenced by histological changes and a decrease in collagen accumulation. SGE treatment also led to a reduction in markers of HSC activation and inflammation as well as an improvement in blood biochemical parameters. Furthermore, SGE administration diminished fibrotic responses following acute liver injury. Mechanistically, SGE treatment prevented HSC activation and inhibited the phosphorylation and nuclear translocation of Smad2/3, which are induced by transforming growth factor (TGF)-ß1 in HSCs. Our findings indicate that SGE exhibits anti-fibrotic effects by inhibiting TGFß1-Smad2/3 signaling in HSCs.

Engineering of a bacterial outer membrane vesicle to a nano-scale reactor for the biodegradation of ß-lactam antibiotics.

Bacterial outer membrane vesicles (OMVs) are small unilamellar proteoliposomes, which are involved in various functions including cell to cell signaling and protein excretion. Here, we have engineered the OMVs of Escherichia coli to nano-scaled bioreactors for the degradation of ß-lactam antibiotics. This was exploited by targeting a ß-lactamase (i.e., CMY-10) into the OMVs of a hyper-vesiculating E. coli BL21(DE3) mutant. The CMY-10-containing OMVs, prepared from the E. coli mutant cultures, were able to hydrolyze ß-lactam ring of nitrocefin and meropenem to a specific rate of 6.6 × 10-8 and 3.9 × 10-12 µmol/min/µm3 of OMV, which is approximately 100 and 600-fold greater than those of E. coli-based whole-cell biocatalsyts. Furthermore, CMY-10, which was encapsulated in the engineered OMVs, was much more stable against temperature and acid stresses, as compared to free enzymes in aqueous phase. The OMV-based nano-scaled reaction system would be useful for the remediation of a variety of antibiotics pollution for food and agricultural industry.

Microporous polymers with cascaded cavities for controlled transport of small gas molecules.

In membrane-based separation, molecular size differences relative to membrane pore sizes govern mass flux and separation efficiency. In applications requiring complex molecular differentiation, such as in natural gas processing, cascaded pore size distributions in membranes allow different permeate molecules to be separated without a reduction in throughput. Here, we report the decoration of microporous polymer membrane surfaces with molecular fluorine. Molecular fluorine penetrates through the microporous interface and reacts with rigid polymeric backbones, resulting in membrane micropores with multimodal pore size distributions. The fluorine acts as angstrom-scale apertures that can be controlled for molecular transport. We achieved a highly effective gas separation performance in several industrially relevant hollow-fibrous modular platform with stable responses over 1 year.

Endoplasmic Reticulum Stress Increases DUSP5 Expression via PERK-CHOP Pathway, Leading to Hepatocyte Death.

Hepatocyte death is critical for the pathogenesis of liver disease progression, which is closely associated with endoplasmic reticulum (ER) stress responses. However, the molecular basis for ER stress-mediated hepatocyte injury remains largely unknown. This study investigated the effect of ER stress on dual-specificity phosphatase 5 (DUSP5) expression and its role in hepatocyte death. Analysis of Gene Expression Omnibus (GEO) database showed that hepatic DUSP5 levels increased in the patients with liver fibrosis, which was verified in mouse models of liver diseases with ER stress. DUSP5 expression was elevated in both fibrotic and acutely injured liver of mice treated with liver toxicants. Treatment of ER stress inducers enhanced DUSP5 expression in hepatocytes, which was validated in vivo condition. The induction of DUSP5 by ER stress was blocked by either treatment with a chemical inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, or knockdown of C/EBP homologous protein (CHOP), whereas it was not affected by the silencing of IRE1 or ATF6. In addition, DUSP5 overexpression decreased extracellular-signal-regulated kinase (ERK) phosphorylation, but increased cleaved caspase-3 levels. Moreover, the reduction of cell viability under ER stress condition was attenuated by DUSP5 knockdown. In conclusion, DUSP5 expression is elevated in hepatocytes by ER stress through the PERK-CHOP pathway, contributing to hepatocyte death possibly through ERK inhibition.

Determination of plutonium isotopes (238,239,240Pu) and strontium (90Sr) in seafood using alpha spectrometry and liquid scintillation spectrometry.

The present study was carried out to survey the levels of plutonium isotopes (238,239,240Pu) and strontium (90Sr) in domestic seafood in Korea. In current, regulatory authorities have analyzed radionuclides, such as 134Cs, 137Cs and 131I, in domestic and imported food. However, people are concerned about contamination of other radionuclides, such as plutonium and strontium, in food. Furthermore, people who live in Korea have much concern about safety of seafood. Accordingly, in this study, we have investigated the activity concentrations of plutonium and strontium in seafood. For the analysis of plutonium isotopes and strontium, a rapid and reliable method developed from previous study was used. Applicability of the test method was verified by examining recovery, minimum detectable activity (MDA), analytical time, etc. Total 40 seafood samples were analyzed in 2014-2015. As a result, plutonium isotopes (238,239,240Pu) and strontium (90Sr) were not detected or below detection limits in seafood. The detection limits of plutonium isotopes and strontium-90 were 0.01 and 1 Bq/kg, respectively.

Vibrio vulnificus glycogen branching enzyme preferentially transfers very short chains: N1 domain determines the chain length transferred.

The glycogen branching enzyme from Vibrio vulnificus (VvGBE) transfers short side chains (DP 3-5) significantly greater than any other bacterial glycogen branching enzyme (GBE). To elucidate the role of the N-domain of VvGBE in the unique branching pattern, domain-truncated (N1 and N) and N1-domain-swapped (with VvGBE N1 replacing the counter part of Escherichia coli GBE) mutants were constructed. The truncation mutants synthesized branched products with a greatly reduced proportion of short chains. The swapping mutant exhibited a branching pattern of the short chain region similar to that of VvGBE. We conclude that the N1-domain of VvGBE has a crucial role in the determination of the branching pattern of glycogen.

Simultaneous microfabrication and tuning of the permselective properties in microporous polymers using X-ray lithography.

Microchannels are fabricated using a photosensitive polymer to which microporosity is tuned with different X-ray doses. Using hard X-ray irradiation, the micropattern is positioned with various geometries in a multi-level, three-dimensional structure, while controlling the pore size and transport properties of small molecules. This highly reliable fabrication process has potential for use in microfluidic devices with enhanced transport properties through microchannels.