Search

The authors have requested that this preprint be removed from Research Square.

Saccharomyces cerevisiae is an important model eukaryotic microorganism and widely applied in fundamental research and the production of various chemicals. Its ability to efficiently and precisely control the expression of multiple genes is valuable for metabolic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)-mediated regulation enables complex gene expression programming; however, the regulation efficiency is often limited by the efficiency of pertinent regulators. Here, we developed CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression in S. cerevisiae. By introducing protein scaffolds (SPY and SunTag systems) to recruit multiple copies of regulators to different nuclease-deficient CRISPR proteins and design optimization, our system amplified gene regulation efficiency significantly. The gene activation and repression efficiencies reached as high as 34.9-fold and 95%, respectively, being 3.8- and 8.6-fold higher than those observed on the direct fusion of regulators with nuclease-deficient CRISPR proteins, respectively. We then applied the orthogonal bifunctional CRISPR-mediated transcriptional regulation system to regulate the expression of genes associated with 3-hydroxypropanoic acid production to deduce that CRISPR-associated regulator recruiting systems represent a robust method for simultaneously regulating multiple genes and rewiring metabolic pathways.

An exploration study was conducted to explore what Chinese undergraduates considered to be a “good job.” A total of 143 ( M = 21.45, SD = 1.53, 51.04% male) undergraduates from 10 cities participated in this study. The prototype research methodology was applied to explore the underlying complex structure of the “good job.” The results revealed 157 items associated with the “good job” that were merged into two categories “high-quality work” and “high-quality life.” Furthermore, each category included basic- and superordinate-levels prototypes. The structure depicted Chinese undergraduates’ expectations of a “good job” and emphasized the importance of work-life balance among young people. Additionally, traditional Chinese culture was consistently found to have a significant impact on young people’s expectations of a “good job.” These findings have implications for career development research and career counseling practices about Chinese young people.

Supplementary Table 1. Relative miRIP isolation of miRNAs predicted by TargetScan in HepG2 and PC-3 cells.

microRNA (miRNA) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, called miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs that bound p21 mRNA in HepG2 and PC-3 cells were identified by the miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA, both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21, which is related to poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by upregulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs that target STAT3 mRNA were also identified by the miRIP method in HepG2 cells. Our results demonstrated that the miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs that target specific mRNA in vivo. Cancer Res; 75(14); 2875–85. ©2015 AACR.

New P,Nsp3 bidentate ligands containing two chiral carbon centers were developed and applied to palladium-catalyzed asymmetric allylic substitution reactions. Good generalities with various nucleophiles, including carbon, nitrogen and oxygen containing...

To know about the reasons leading to variations in dust control efficiency of the surfactant solution spray on coal dust (from the same coal source) with different diameters, the changes of coal dust surface features (specific surface area, pore volume, gas adsorption, and surface potential) with crush degrees and their effects on the wettability were investigated. The experimental results indicated that the surface characteristics of coal dust showed remarkably positive correlations with the crush degree. For example, dust size was reduced from 114.96 to 18.71 μm, the pore volume and gas adsorption of coal dust surface was enhanced by 75%, 104.5%, respectively. It made gas film around dust particles more easily been generated, hindering the contact between dust particles and droplets. The adsorption rate of the surfactant molecules on the coal dust surface was significantly reduced with the dust size decreased, increasing the difficulty of capturing coal dust by surfactant solution. Additionally, based on the linear fitting analysis between surface features and the dust control efficiency, it was indicated that the increased gas adsorption and pore structures on the dust surface was the key factors weakening the dust removal efficiency of the surfactant solution from the perspective of the physical features of coal dust. This study was conducive to optimizing the surfactant-aided dust control technology to better capture coal dust with small size.

The oleaginous yeast Yarrowia lipolytica represents an environmentally friendly platform cell factory for β-carotene production. However, Y. lipolytica is a dimorphic species that can undergo a yeast-to-mycelium transition when exposed to stress. The mycelial form is unfavorable for industrial fermentation. In this study, β-carotene-producing Y. lipolytica strains were constructed via the integration of multiple copies of 13 genes related to the β-carotene biosynthesis pathway. The β-carotene content increased by 11.7-fold compared with the start strain T1. As the β-carotene content increased, the oval-shaped yeast form was gradually replaced by hyphae, implying that the accumulation of β-carotene in Y. lipolytica induces a morphological transition. To relieve this metabolic stress, the strains were morphologically engineered by deleting CLA4 and MHY1 genes to convert the mycelium back to the yeast form, which further increased the β-carotene production by 139%. In fed-batch fermentation, the engineered strain produced 7.6 g/L and 159 mg/g DCW β-carotene, which is the highest titer and content reported to date. The morphological engineering strategy developed here may be useful for enhancing chemical synthesis in dimorphic yeasts.

Conjugated dienes are versatile building blocks and prevalent substructures in synthetic chemistry. Herein, we report a method for the stereoselective hydroalkenylation of alkynes, utilizing readily available enol triflates. We leveraged an in situ generated and geometrically pure vinyl-Cu(I) species to form the Z,Z- or Z,E-1,3-dienes in excellent stereoselectivity and yield. This approach allowed for the synthesis of highly substituted Z-dienes, including pentasubstituted 1,3-dienes, which are difficult to prepare by existing approaches.

The Sn + H2O reaction is important in both hydrogen production through solar thermochemical redox cycles and investigations like the treatment of nuclear reactors or flame inhibition. Based on single molecule model, this work systematically explores its possible reacting channels in different spin multiplicities at CCSD(T)//DFT level of theory to reveal the underlined mechanism for its reported efficient hydrogen production. It is found that the singlet and triplet potential energy surfaces cross each other during the water attacking process, which makes the hydrogen production channel in the singlet state energetically favored. Quantitative calculations about the possibility of surface crossing and spin inversion with respect to minimum energy crossing point, spin-orbit coupling coefficient and intersystem crossing probability confirm that the optimal reacting pathway involves the two-state reaction scenario. This special reactive pattern makes hydrogen production not only possible but also efficient. Analysis of the equilibrium constant of reactive channels and their variation with temperature reveals the performance of two-state reaction channel agrees well with reported data range and nontrivial temperature dependence.

The lane lines’ length, width, and direction are very regular, serialized, and structurally associated, which are not easily affected by the environment. To enhance lane detection in a complicated environment, an approach combines visual information with the spatial distribution. Firstly, the grid density of the target detection algorithm YOLOv3 (you only look once V3) is improved from S×S to S×2S, aiming at the particular points in the bird’s-eye view where the lane lines had different densities in the horizontal and vertical directions. The obtained YOLOv3 (S×2S) is more suitable for detecting objects with small and large aspect ratios. It also identifies image features along with balances the detection speed and accuracy. Secondly, based on a bi-directional gated recurrent unit (BGRU), a new lane line prediction model BGRU-Lane (BGRU-L) based on the distribution of lane lines is proposed using the characteristic of lane line serialization and structural correlation. Finally, Dempster-Shafer (D-S) algorithm based on confidence was used to integrate the results of YOLOv3 (S×2S) and BGRU-L to improve the lane line detection ability under complex environments. The experiment was carried out on the Karlsruhe Institute of Technology and Toyota Technological Institute (KITTI) dataset, while Euro Truck Simulator 2 (ETS2) is used as a supplement dataset. After fusing YOLOv3 (S×2S) and BGRU-L models in the D-S model, the detection results have high accuracy in a complex environment by 90.28 mAP. The detection speed is 40.20fps, which enables real-time detection.

While there has been research focused on interpersonal relationships and their impact on stress and well-being, no instrument has been developed to comprehensively evaluate interpersonal stressors. This research sought to develop and validate an Interpersonal Stressors Scale (ISS) for Chinese college students through three studies. Focus groups were used to generate the initial item pool (Study 1). Then two large samples ( N1 = 511; N2 = 330) were collected to explore the factor structure of the ISS and subsequently examine its reliability and validity estimates (study 2 and 3). Initial results indicated a model with 27 items and five first-order factors (interaction difficulty, behaving as expected, social criticism, relationship maintenance, and indebtedness avoidance) as well as two second-order factors (self-imposed stressors and other-imposed stressors) with strong psychometric properties. Criterion-related validity estimates indicated these two kinds of stressors were both associated with stress while having different relationships with general anxiety, depression, social anxiety, interpersonal satisfaction, and self-efficacy in social interactions. The nature and function of the structure for the ISS were discussed as well as the practical and research implications.

Producing high value-added products from waste lipid feedstock by microbial cell factory has great advantages to minimize the pollution as well as improve the economic value of wasted oils and fats. Yarrowia lipolytica is a non-conventional oleaginous yeast and can grow on a variety of hydrophobic substrates. In this study, we explored its ability to synthesize α-farnesene, an important sesquiterpene, using lipid feedstock. Based on the α-farnesene production strain, we constructed previously, we identified that Erg12 was the key limiting factor to further increase the α-farnesene production. The α-farnesene production was improved by 35.8% through increasing the copy number of ERG12 and FSERG20 on oleic acid substrate. Expression of heterologous VHb further improved α-farnesene production by 12.7%. Combining metabolic engineering with the optimization of fermentation conditions, the α-farnesene titer and yield reached 10.2 g/L and 0.1 g/g oleic acid, respectively, in fed-batch cultivation. The α-farnesene synthesis ability on waste cooking oil and other edible oils were also explored. Compared with using glucose as carbon source, using lipid substrates obtained higher α-farnesene yield and titer, but lower by-products accumulation, demonstrating the advantage of Y. lipolytica to synthesize high value-added products using lipid feedstock.

β-Carotene is a provitamin A precursor and an important antioxidant that is used widely in the aquaculture, food, cosmetic, and pharmaceutical industries. Oleaginous Yarrowia lipolytica has been demonstrated as a competitive producer microorganism for the production of hydrophobic β-carotene through rational engineering strategies. However, the limited understanding of the complexity of the metabolic network between carotenoid biosynthesis and other cellular processes has hampered further advancement. Genome-scale mutagenesis and high-throughput screening of mutagenesis libraries have been extensively employed in gene mining or in the identification of key targets associated with particular phenotypes. In this study, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed. Here, we used a flow cytometry approach to improve screening efficiency and eliminate the interference of intermediate metabolites. The targets obtained in this study can be used in studies focusing on metabolic engineering in the future for improving carotenoid production. IMPORTANCE β-Carotene is a high-value-added product that is widely used in the aquaculture, food, cosmetic, and pharmaceutical industries. In our previous study, Yarrowia lipolytica has been engineered extensively to produce β-carotene. To further improve its production, high-throughput screening and the identification of new beneficial gene targets are required. Herein, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed.

NaYF