Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production

Phloroglucinol synthase PhlD is a type III polyketide synthase capable of directly converting three molecules of malonyl-CoA to an industrially important chemical—phloroglucinol (1, 3, 5-trihydroxylbenzene). Although this enzymatic process provides an attractive biosynthetic route to phloroglucinol, the low productivity of PhlD limits its further practical application. Here we used protein engineering coupled with in situ product removal to improve the productivity of phoroglucinol biosynthesis in recombinant Escherichia coli. Specifically, directed evolution was used to obtain a series of thermostable PhlD mutants with the best one showing over 24-fold longer half-life of thermal inactivation than the wild-type enzyme at 37 °C. When introduced into a malonyl-CoA overproducing E. coli strain, one of the mutants showed 30 % improvement in phloroglucinol productivity compared to the wild-type enzyme in a shake-flask study and the final phloroglucinol concentration reached 2.35 g/L with 25 % of theoretical yield. A continuous product extraction strategy was designed to remove the toxic phloroglucinol product from the cell media, which further increased the titer of phloroglucinol to 3.65 g/L, which is the highest phloroglucinol titer ever reported to date.     

Microbial Interaction Network Estimation via Bias-Corrected Graphical Lasso

With the increasing availability of microbiome 16S data, network estimation has become a useful approach to studying the interactions between microbial taxa. Network estimation on a set of variables is frequently explored using graphical models, in which the relationship between two variables is modeled via their conditional dependency given the other variables. Various methods for sparse inverse covariance estimation have been proposed to estimate graphical models in the high-dimensional setting, including graphical lasso. However, current methods do not address the compositional count nature of microbiome data, where abundances of microbial taxa are not directly measured, but are reflected by the observed counts in an error-prone manner. Adding to the challenge is that the sum of the counts within each sample, termed “sequencing depth,” is an experimental technicality that carries no biological information but can vary drastically across samples. To address these issues, we develop a new approach to network estimation, called BC-GLASSO (bias-corrected graphical lasso), which models the microbiome data using a logistic normal multinomial distribution with the sequencing depths explicitly incorporated, corrects the bias of the naive empirical covariance estimator arising from the heterogeneity in sequencing depths, and builds the inverse covariance estimator via graphical lasso. We demonstrate the advantage of BC-GLASSO over current approaches to microbial interaction network estimation under a variety of simulation scenarios. We also illustrate the efficacy of our method in an application to a human microbiome data set.

     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

Whole‐brain microcirculation detection after ischemic stroke based on swept‐source optical coherence tomography

The occurrence and development of ischemic stroke are closely related to cerebral blood flow. Real‐time monitoring of cerebral perfusion level is very useful for understanding the mechanisms of the disease. A wide field of view (FOV) is conducive to capturing lesions and observing the progression of the disease. In this paper, we attempt to monitor the whole‐brain microcirculation in middle cerebral artery occlusion (MCAO) rats over time using a wide FOV swept‐source OCT (SS‐OCT) system. A constrained image registration algorithm is used to remove motion artifacts that are prone to occur in a wide FOV angiography. During ischemia, cerebral perfusion levels in the left and right hemispheres, as well as in the whole brain were quantified and compared. Changes in the shape and location of blood vessels were also recorded. The results showed that the trend in cerebral perfusion levels of both hemispheres was highly consistent during MCAO, and the position of the blood vessels varied over time. This work will provide new insights of ischemic stroke and is helpful to assess the effectiveness of potential treatment strategies.      

Single,Double and Quadruple Alanine Substitutions at Oligomeric Interfaces Identify Hydrophobicity as the Key Determinant of Human Neutrophil Alpha Defensin HNP1 Function

HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr¹⁶, Ile²⁰, Leu²⁵, and Phe²⁸. Previously, alanine scanning mutagenesis identified each of these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions.     

Identification of a Novel Universal Potential Epitope on the Cytoplasmic Tail of H7N9 Virus Hemagglutinin

<正>Dear Editor,H7 N9 is a recently identified subtype of influenza A virus that caused a major outbreak in humans in China in 2013.According to the latest data provided by the Chinese Center for Disease Control and Prevention(http://www.moh.gov.cn/zwgk/yqbb3/ejlist.shtml, updated on October 31, 2018),the mortality rate of H7 N9 infections in China amounts to     

普洱熟茶后发酵优势菌臭曲霉的生物学特性

为了开发普洱熟茶生产的规范技术,本文对普洱茶后发酵优势芮之一的臭曲霉的生物学特性进行了研究.结果表明,该菌株对酸碱度有广幅的适应性;在以硫酸铵或豆饼粉为氮源,玉米粉或果糖为碳源的培养基中生长迅速;培养温度以30℃最为适宜.同时,对菌落的牛长规律及形态特征进行了观察与分析.研究结果为该菌株的大量培养,以及普洱茶的规范化生产技术提供了基础.     

LDL coating pVEGF/polyethylenimine complex enhances vascular endothelial growth factor expression

The major limitations to non-viral gene delivery are relatively low efficiency and cytotoxicity, which need to be addressed in the design of new vectors. In this study, negatively charged low density lipoproteins (LDL) were coated onto positively charged pVEGF/PEI complexes to form pVEGF/PEI/LDL terplexes by a two-step procedure. The biocompatible LDL was introduced to reduce the cytotoxicity of the gene delivery system and increase its affinity to cells. The successful formation of pVEGF/PEI/ LDL terplexes was confirmed by their near-neutral and slightly negative surface charges. The pVEGF/PEI/LDL terplexes were well-defined sub-micron spherical particles. On the cell viability assay, both of the PEI/LDL combined vector and pVEGF/PEI/LDL terplexes exhibited much lower cytotoxicity to HeLa cells and HUVE cells than those of PEI and pVEGF/PEI complexes, attributed to the shielding effect of the LDL. pEGFP/PEI/LDL terplexes showed significantly higher transfection efficiency in comparison to pEGFP/PEI complexes in serum-containing medium. pVEGF/PEI/LDL terplexes at their optimal N/P ratio and LDL/PEI weigh ratio induced higher expression levels of VEGF protein in HUVE cells than those of pVEGF/PEI complexes. Therefore, the pVEGF/PEI/LDL terplexes could be used as a promising gene delivery system to enhance VEGF protein expression.