[Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum].

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 µmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

On-Surface Decarboxylation Coupling Facilitated by Lock-to-Unlock Variation of Molecules upon the Reaction.

On-surface synthesis (OSS) involving relatively high energy barriers remains challenging due to a typical dilemma: firm molecular anchor is required to prevent molecular desorption upon the reaction, whereas sufficient lateral mobility is crucial for subsequent coupling and assembly. By locking the molecular precursors on the substrate then unlocking them during the reaction, we present a strategy to address this challenge. High-yield synthesis based on well-defined decarboxylation, intermediate transition, and hexamerization is demonstrated, resulting in an extended and ordered network exclusively composed of the newly synthesized macrocyclic compound. Thanks to the steric hindrance of its maleimide group, we attain a preferential selection of the coupling. This work unlocks a promising path to enrich the reaction types and improve the coupling selectivity hence the structual homogeneity of the final product for OSS.

Long-range ordered and atomic-scale control of graphene hybridization by photocycloaddition.

Chemical reactions that convert sp2 to sp3 hybridization have been demonstrated to be a fascinating yet challenging route to functionalize graphene. So far it has not been possible to precisely control the reaction sites nor their lateral order at the atomic/molecular scale. The application prospects have been limited for reactions that require long soaking, heating, electric pulses or probe-tip press. Here we demonstrate a spatially selective photocycloaddition reaction of a two-dimensional molecular network with defect-free basal plane of single-layer graphene. Directly visualized at the submolecular level, the cycloaddition is triggered by ultraviolet irradiation in ultrahigh vacuum, requiring no aid of the graphene Moiré pattern. The reaction involves both [2+2] and [2+4] cycloadditions, with the reaction sites aligned into a two-dimensional extended and well-ordered array, inducing a bandgap for the reacted graphene layer. This work provides a solid base for designing and engineering graphene-based optoelectronic and microelectronic devices.

Prognostic value of neutrophil-to-lymphocyte ratio in sepsis: A meta-analysis.

BACKGROUND: Neutrophil-to-lymphocyte ratio (NLR) has been used to predict the prognosis of patients with sepsis with inconsistent results. This meta-analysis aimed to clarify the prognostic value of NLR in patients with sepsis. METHODS: A comprehensive literature search for relevant studies, published prior to March 2019, was conducted using PubMed, Web of Science, and the China National Knowledge. Infrastructure database. Standard mean differences (SMDs) with 95% confidence intervals (CI) were used to evaluate the NLR of patients with sepsis retrospectively. Hazard ratios (HRs) with 95% CIs were used to evaluate the prognostic value of NLR in patients with sepsis. RESULTS: Patients from 14 studies (n = 11,564) were selected for evaluation. Nine studies (1371 patients) analyzed the NLR in these patients. The pooled results showed significantly higher NLR in non-survivors than in survivors (random-effects model: SMD = 1.18, 95% CI; 0.42-1.94). Nine studies (10,685 patients) evaluated the prognostic value of NLR for sepsis; the pooled results showed that higher NLR was associated with poor prognosis in patients with sepsis (fixed-effects model: HR = 1.75, 95% CI; 1.56-1.97). Subgroup analysis revealed that study design, cut-off NLR, or primary outcome did not affect the prognostic value of NLR in patients with sepsis. CONCLUSION: This meta-analysis indicates that NLR may be a helpful prognostic biomarker of patients with sepsis and that higher NLR values may indicate unfavorable prognoses in these patients.

Activating transcription factor 4 is required for high glucose inhibits proliferation and differentiation of MC3T3-E1 cells.

Activating transcription factor 4 (ATF4) promotes bone formation in human bone marrow mesenchymal stem cells. However, the underlying mechanisms of ATF4 in high glucose-induced injury of osteoblast still remain unclear. Small interfering RNA and plasmid targeting ATF4 were used to transfect MC3T3-E1 cells to knock down and overexpress ATF4 using Lipofectamin 3000. Cell viability, alkaline phosphatase (ALP) activity and levels were determined by MTT, ALP kit assay, quantitative real-time (qRT)-PCR and Western blot. Osteocalcin (OCN) expression was determined by ELISA, PCR and Western blot. The mRNA and protein levels of ATF4, glucose regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) were detected by PCR and Western blot. In the current study, viabilities of MC3T3-E1 cells were inhibited by high glucose. Meanwhile, the mRNA and protein levels of ATF4 were effectively up-regulated in high glucose-incubated MC3T3-E1 cells. By conducting functional experiments, silencing ATF4 induced by small interfering RNA partially reversed the inhibitory effects of high glucose on viabilities of MC3T3-E1 cells. We also found that the expressions of ER stress-related proteins (ATF4, GRP78 and CHOP) were higher in high glucose-treated MC3T3-E1 cells but were inhibited by siATF4. However, overexpression of AFT4 had opposite results, and high glucose attenuated the protein levels of osteogenic marker genes ALP and OCN, which were further inhibited by ATF4 knockout gene. Thus, ATF4 was a necessary gene for high glucose to inhibit the proliferation and differentiation of MC3T3-E1 cells.

Enabling Lithium-Metal Anode Encapsulated in a 3D Carbon Skeleton with a Superior Rate Performance and Capacity Retention in Full Cells.

Suppressing the formation of lithium (Li) dendrites is central to implementing Li-metal anode, which has gained growing attention due to its ultrahigh specific capacity and low redox potential. Here, a novel approach is adopted to deposit Li-metal within a rigid three-dimensional (3D) carbon paper (3DCP) network, which consists of a cross-link framework of carbon fibers and graphene nanosheets (GNs). This unique structure yields a uniform distribution of Li-nuclei during the preliminary stage of Li-plating and the formation of a stable solid-electrolyte interface. The as-obtained anode can deliver a high areal capacity of 10 mAh cm-2 without the dendritic formation after 1000 cycles in a Li@3DCP/LiFePO4 full cell at 4 C. In addition, the Li@3DCP anode displays low voltage platform (<20 mV at 1 mA cm-2), high plating/stripping efficiency (99.0%), and long lifespan (>1000 h). When coupled with LiNi0.8Co0.15Al0.05O2 cathode, the Li@3DCP electrode exhibits a superior rate capability up to 10 C and high temperature performance (60 °C). The unprecedented performance is attributed to the desirable combination of micro/nanostructures in 3DCP, in which carbon fiber framework provides the mechanical stability for volume change, whereas numerous lithiophilicity sites on GNs enable the suppression of Li-dendrite growth.