Enhancing acid tolerance of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> with glutathione

Leuconostoc mesenteroides is a commercially important lactic acid bacterium currently used as a starter for kimchi and kefir. However, its sensitivity to acid stress limits its performance. L. mesenteroides was grown in a medium supplemented with 3.2 or 6.4 mM glutathione (GSH), and cell survival rates were measured during a long-term mild acid challenge (pH 4.0). As a result, GSH was imported by the cells and protected against acid stress; thereafter it was consumed as a nutrient. Acid stress resistance of starter cultures of this bacterium can thus be improved by cultivating it in media supplemented with GSH.     

Boosting the free fatty acid synthesis of <Emphasis Type="Italic">Escherichia coli</Emphasis> by expression of a cytosolic <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> thioesterase

Background

Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.

Results

We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser¹⁰, Gly⁴⁸, Asn⁷⁷, Asp¹⁵⁸ and His¹⁶¹ residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.

Conclusions

For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.

     

诱导突变高粱愈伤组织初探

本实验以突变体高粱的不同外植体 ,成熟种子、成熟胚、茎尖、和幼胚等为材料 ,诱导愈伤组织。经出愈率和生长状况观察 ,幼胚最好 ,成熟胚较好 ,成熟种子和茎尖略差。对于茎尖 ,2 ,4 D与KT搭配较好 ;成熟胚而言 ,不加细胞分裂素 ,加适量 2 ,4 D浓度效果较好 ,幼胚愈伤组织诱导和培养不需要细胞分裂素 ,加浓度为 2 .0mg/L2 ,4 D效果较好 ;若加KT ,同时要提高 2 ,4 D浓度 ;成熟种子培养 ,需要细胞分裂素     

Liquid chromatography-tandem mass spectrometric method for determination of salivary 17alpha-hydroxyprogesterone: a noninvasive tool for evaluating efficacy of hormone replacement therapy in congenital adrenal hyperplasia

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the quantification of 17alpha-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC-MS-MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-mul sample, and the limit of quantitation was 5.0 pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive (r(2)=0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy.     

人Ⅱ型囊泡单胺转运体基因的克隆

目的 为获得人Ⅱ型囊泡单胞转运体（vesicular monomine transporter-2VMAT2)以治疗帕金森病,克隆添加Kozak序列的VMAT2cDNA,用于真核细胞表达。方法 从人胎脑中提取总RNA,逆转录成cDNA,设计一对引物用PCR方法扩增目的cDNA片段并将其重级一起pGEM-Easy-T载体中,酶切鉴定插入片段后,进行全序列测定。结果 RT-PCR扩增到1637bp的带有Kozak序列的cDNA片段。结论 克隆人VMAT2cDNA片断,可用于真核细胞表达。     

Synergistic Regulation of Intrinsic Lithiophilicity and Mass Transport Kinetics of Non-Lithium-Alloying Nucleation Sites for Stable Operation of Low N/P Ratio Lithium Metal Batteries

Constructing functional materials on a 3D host is an efficient strategy to tackle issues of lithium (Li) metal anodes. Although non-Li-alloying materials provide structural stability during cycling due to reduced lattice distortions, low lithiophilicity and sluggish mass transport kinetics limit their functionality. Herein, a synergistic strategy is proposed to improve intrinsic lithiophilicity and mass transport kinetics of non-Li-alloying nucleation sites and demonstrate its remarkable efficacy. Two carbon fiber (CF) hosts coated by non-Li-alloying nanosheets with and without oxygen-enriched carbon filler (OCF) as lithiophilicity and mass transport booster (OCF-DSC@CF and DSC@CF, respectively) are constructed and their physiochemical properties are systematically evaluated to reveal the efficacy of OCF. By advanced characterization techniques, including 3D tomography and location-dependent electron energy loss spectroscopies, the complex heterostructure of OCF-DSC@CF with distinctive roles of each constituent is clearly identified. As verified by theoretical and electrochemical analyses, the incorporation of OCF endows OCF-DSC@CF with substantially improved lithiophilicity and mass transport kinetics. Moreover, OCF-DSC@CF induces a multifunctional SEI enriched with LiF and LiC_x, which exhibits well-balanced electrical resistivity and ionic conductivity. Benefiting from these attributes, OCF-DSC@CF exhibits an unprecedented cyclability under a low N/P ratio of 1.8, achieving 700 cycles at 0.5C with an exceptional capacity retention of 97.8%.     

基于转录组测序的油菜花粉BpFLS基因克隆与分析

【目的】青海门源油菜耐寒冷、抗性强,其花粉积累了丰富的类黄酮物质,该研究可为提高黄酮醇含量的基因工程育种提供参考依据。【方法】研究采用转录组测序技术从青海门源油菜花粉和河南郑州油菜花粉筛选差异表达的黄酮醇合成酶基因,并对BpFLS基因进行生物信息学分析和RACE-PCR克隆。【结果】(1)筛选出6个差异表达的油菜花粉黄酮醇合成酶基因,BpFLS1-1基因在青海门源油菜花粉中差异最大且表达上调。(2)BpFLS1-1基因编码的氨基酸序列具有黄酮醇合成酶活性和黄烷酮-3-羟化酶活性。(3)BpFLS1-1基因可能含有新的蛋白质编码区序列,其长度为1 170 bp。【结论】BpFLS1-1基因在门源油菜花粉积累丰富的类黄酮物质及油菜抗逆性方面发挥重要功能。     

Human umbilical cord mesenchymal stem cell-derived mitochondria (PN-101) attenuate LPS-induced inflammatory responses by inhibiting NFκB signaling pathway

Inflammation is one of the body’s natural responses to injury and illness as part of the healing process. However, persistent inflammation can lead to chronic inflammatory diseases and multi-organ failure. Altered mitochondrial function has been implicated in several acute and chronic inflammatory diseases by inducing an abnormal inflammatory response. Therefore, treating inflammatory diseases by recovering mitochondrial function may be a potential therapeutic approach. Recently, mitochondrial transplantation has been proven to be beneficial in hyperinflammatory animal models. However, it is unclear how mitochondrial transplantation attenuates inflammatory responses induced by external stimuli. Here, we isolated mitochondria from umbilical cord-derived mesenchymal stem cells, referred as to PN-101. We found that PN-101 could signifi-cantly reduce LPS-induced mortality in mice. In addition, in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 macrophages, PN-101 attenuated LPS-induced increase production of pro-inflammatory cytokines. Furthermore, the anti-inflammatory effect of PN-101 was mediated by blockade of phosphorylation, nuclear translocation, and trans-activity of NFκB. Taken together, our results demonstrate that PN-101 has therapeutic potential to attenuate pathological inflammatory responses.     

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.