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In this work, the solubilities of carbamazepine (CBZ) (form III) in ethyl acetate, methyl acetate, ethylene glycol, chloroform and cyclohexylamine were determined by laser monitoring techniques at pressure above sea level, and the solubility data of CBZ (form III) in different pure solvents were fitted by the Modified Apelblat model and λh model. The result shows that the solubility of CBZ (form III) in five solvents increases as temperature rises, and the solubility in chloroform was the largest. The experimental solubility values of CBZ (form III) in ethyl acetate, methyl acetate, chloroform and cyclohexylamine were in better agreement with the simulated fitting values of the λh model. For ethylene glycol, the σ value was much larger than the other four solvents, and it can be seen from the λh model that ethylene glycol was closer to the ideal solution system than the other four solvents.

In this work, ternary phase diagrams of telmisartan-saccharin (TEL-SAC) cocrystal in methanol, ethanol, and quasi-ternary sections of the quaternary system TEL-SAC–chloroform–methanol were determin...

In this work, solubility of exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETA) was measured and discussed in acetone + ethanol, methyl acetate + ethanol, 1,4-dioxane + ethanol, and ethyl acet...

Limited by performance of hardware, electronic displays cannot reach the actual brightness of glowing objects, but the method of using glare illusions to simulate real light sources has been widely studied. These studies mainly focused on exploring the influence of gradients on brightness perception. As one of the basic perception characteristics of light sources, it is still unknown whether radial streaks will affect people's perception of light sources. Based on previous researches, we propose a new rendering model of self-luminance objects, which can effectively simulate the radial streaks formed by diffraction of light. By a Maximum Difference Scaling experiment, the influence of streak intensity, luminance gradient and streak fusion state on the fidelity of the light source is further explored. We found that streak intensity and fusion state can effectively improve the realism of virtual light sources.

In recent years, hybrid rocking columns have drawn more and more interests from researchers, due to their self-centering capacity. The energy-dissipation bars, which are generally applied at the rocking joints of hybrid rocking columns, could improve their energy dissipation capacities. Thus, the reasonable reinforcement ratio of energy-dissipation bars is much required for engineering applications. To determine the reasonable reinforcement ratio of energy-dissipation bars, a numerical investigation is conducted in this paper based on nonlinear time-history analysis. The analysis results show that a reasonable reinforcement ratio of energy-dissipation bars can effectively reduce seismic displacements of the hybrid rocking columns, without excessive residual deformations. Further, the reasonable reinforcement ratio of energy-dissipation bars for hybrid rocking columns with different periods is proposed in this paper.

The rocking column is a kind of excellent seismic-resistant and resilient structure. However, during the rocking process, local pressure on the rocking interface may result in spalling and crushing of concrete at the toe. In order to reduce the local damage of the rocking interface, as well as accelerate the connection between prefabricated column and footing, a kind of mechanical connection device (MCD) was proposed in this research. The MCD is used at the bottom of column and ofters rapid connection of the column to the footing. The recentering force of MCD column is provided by PT strands and axial force of superstructure, and the energy dissipation is provided by U-shaped dissipaters. A simplified analytical method based on interface section analysis is proposed to study the hysteretic behavior of column with MCD. The results suggest that the MCD column has stable hysteretic behavior and can achieve small residual drift after earthquakes by appropriate design.

We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

Because of the long lifetime of newbuilding vessels, shipowners' emission abatement solutions will have a profound impact on the greening transition of the shipping industry. A multinominal logit model is developed to identify the key determinants that affect the shipowners' choice of the solutions. New findings include: (1) Alternative fuels (e.g. liquefied natural gas) are the most attractive choice for gas ships and pure car carriers; and dry bulk ships and crude tankers prefer to use low-sulfur fuels. (2) Singaporean and Japanese shipowners are more likely to adopt the alternative fuels. (3) The IMO 2020 cap has significantly affected shipowners’ preference for scrubbers but not for alternative fuels. It is well known that economic factors affecting a shipowner’s decisions include freight rate and bunker cost but these factors weakly influence the emission abatement solution choice for new ships. This research provides helpful insights for the policymaker to guide shipowners to invest in more eco-friendly vessels and assist various stakeholders in forming efficient decisions.

The concerted action of multiple genes in a time-dependent manner controls complex cellular phenotypes, yet the temporal regulation of gene expressions is restricted on a single-gene level, which limits our ability to control higher-order gene networks and understand the consequences of multiplex genetic perturbations. Here we developed a system for temporal regulation of multiple genes. This system combines the simplicity of CRISPR/Cas9 activators for orthogonal targeting of multiple genes and the orthogonality of chemically induced dimerizing (CID) proteins for temporal control of CRISPR/Cas9 activator function. In human cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. We envision that our system will enable the perturbation of higher-order gene networks with high temporal resolution and accelerate our understanding of gene-gene interactions in a complex biological setting.

SummarySkeletal muscle regeneration is essential for restoring muscle function upon injury and for the maintenance of muscle health with aging. ARHGEF3, a Rho-specific GEF, negatively regulates myoblast differentiation via mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling in a GEF-independent mannerin vitro. Here, we investigated ARHGEF3’s role in skeletal muscle regeneration by creating ARHGEF3 KO mice. These mice exhibited no discernible phenotype under normal conditions. Upon injury, however, ARHGEF3 deficiency enhanced the mass, fiber size and function of regenerating muscles in both young and aged mice. Surprisingly, these effects were not mediated by mTORC2-Akt signaling, but by the GEF activity of ARHGEF3. Furthermore, ARHGEF3 KO promoted muscle regeneration through activation of autophagy, a process that is also critical for maintaining muscle strength. Accordingly, in old mice, ARHGEF3 depletion prevented muscle weakness by restoring autophagy flux. Collectively, our findings identify an unexpected link between ARHGEF3 and autophagy-related muscle pathophysiology.

The solid-liquid phase equilibrium solubility of Doxofylline was measured and discussed in ten pure organic solvents and co-solvent mixtures of acetonitrile + ethanol (+or water) at temperature range from 278.15 to 323.15 K. Results indicated that experimental mole fraction (x) of Doxofylline in ten neat solvents followed this order: acetonitrile > tetrahydrofuran > benzene > acetone > ethyl acetate > methanol > 1-propanol ≈ 2-propanol > ethanol ≈ water, and solubility increased with temperature increasing. Moreover, experimental solubility of Doxofylline firstly increased and then declined with the mass fraction increasing of acetonitrile in acetonitrile + ethanol (+or water) mixture solvents at each temperature. Hansen solubility parameter and solvent effect were used for explaining and discussing solubility rule of Doxofylline in selected solvents. Model correlation indicated the NRTL model was the most suitable than other models to predict solubility of Doxofylline in pure solvents and Sun model was better than other models in mixture solvents with accompanied by temperature and composition. Furthermore, thermodynamic properties of standard molar enthalpy, molar Gibbs free energy and molar entropy in solution was calculated and discussed.

Throughout the biological sciences, the past fifteen years have seen a push towards the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field.

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution.

The solubility values of Doxycycline Hyclate (DH) in water + methanol (or +ethanol and n-propanol) mixture solvents were measured and correlated from 276.95 K to 323.75 K. Experiment results indicated that solubility data in four pure solvents followed this order: methanol> ethanol≈water>n-propanol, and solubility data increased with increasing temperature in all selected solvents. In mixture solvents, the solubility data of Doxycycline Hyclate reached a minimum at xc = 0.75 in water(xc) + methanol(1-xc), a maximum at xc = 0.75 in water(xc) + ethanol(1-xc) and a maximum at xc = 0.8 in water(xc) + n-propanol(1-xc). Moreover, Experimental solubility data was well-correlated with the modified Apelblat model, the Apelblat-Jouyban Acree model and Van't Hoff-Jouyban Acree model, respectively. Correlation coefficient values and relative average deviation values showed these models were very consistent with the experimental values. Furthermore, thermodynamic properties of Doxycycline Hyclate in dissolution process were calculated and discussed from the experimental solubility and parameters of modified Apelblat model.

Transcription activator-like effector nucleases (TALENs) have rapidly emerged as a powerful genome editing tool. The site-specific DNA double-strand breaks generated by TALENs in the human chromosome can induce homologous recombination or non-homologous end joining, resulting in desired genetic modifications. In this study, we report the development of a TALEN variant, SunnyTALEN, with >2.5-fold improved genome editing efficacy in human cells. The corresponding scaffold increases the rate of genetic modification at all the 13 tested loci of human genome and is compatible with heterodimer TALEN architectures. This enhanced and high-efficiency TALEN variant represents a novel second-generation TALEN system and has great potential for biological and therapeutic applications.

Recombinant transcription activator-like effectors (TALEs) have been effectively used for genome editing and gene regulation applications. Due to their remarkable modularity, TALEs can be tailored to specifically target almost any user-defined DNA sequences. Here, we introduce fairyTALE, a liquid phase high-throughput TALE synthesis platform capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency at a material cost of just $5 per TALE. As proof of concept, we synthesized and tested 90 TALEs, each recognizing 27 bp, without restrictions on their sequence composition. 96% of these TALEs were found to be functional, while sequencing confirmation revealed that the nonfunctional constructs were all correctly assembled.

Avian infectious bronchitis virus (IBV) is a member of the group III coronaviruses, which differ from the other groups of coronaviruses in that they do not encode the essential pathogenic factor nonstructural protein 1 (nsp1) and instead start with nsp2. IBV nsp2 is one of the first replicase proteins to be translated and processed in the viral life cycle; however, it has an entirely unknown function. In order to better understand the structural details and functional mechanism of IBV nsp2, the recombinant protein was cloned, overexpressed in Escherichia coli, purified and crystallized. The crystals diffracted to 2.8 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 57.0, b = 192.3, c = 105.7 Å, β = 90.8°. Two molecules were found in the asymmetric unit; the Matthews coefficient was 3.9 Å(3) Da(-1), corresponding to a solvent content of 68.2%.

SARS coronavirus (SARS-CoV) is the aetiological agent of the highly infectious severe acute respiratory syndrome (SARS). To gain a better understanding of SARS-CoV replication and transcription proteins, a preliminary X-ray crystallo­graphic study of the C-terminal domain of SARS-CoV nonstructural protein 2 (nsp2) is reported here. The C-­terminal domain of SARS-CoV nsp2 was cloned, overexpressed, purified and crystallized using polyethylene glycol 5000 monomethyl ether as the precipitant; the crystals diffracted to 2.5 A resolution. The crystals belonged to space group P65, with unit-cell parameters a = b = 112.8, c = 91.1 A, α = β = 90, γ = 120°. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.89 A3 Da−1 and a solvent content of 56.2%.

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized.

Transcription activator-like effector nucleases (TALENs) have rapidly emerged as a powerful genome editing tool. The site-specific DNA double-strand breaks generated by TALENs in the human chromosome can induce homologous recombination or non-homologous end joining, resulting in desired genetic modifications. In this study, we report the development of a TALEN variant, SunnyTALEN, with2.5-fold improved genome editing efficacy in human cells. The corresponding scaffold increases the rate of genetic modification at all the 13 tested loci of human genome and is compatible with heterodimer TALEN architectures. This enhanced and high-efficiency TALEN variant represents a novel second-generation TALEN system and has great potential for biological and therapeutic applications.

Hedgehog (Hh) signaling is fundamentally important for development and adult tissue homeostasis. It is well established that in vertebrates, Sufu directly binds and inhibits Gli proteins, the downstream mediators of Hh signaling. However, it is unclear how the inhibitory function of Sufu toward Gli is regulated. Here we report that the Rusc family of proteins, whose biological functions are poorly understood, form a heterotrimeric complex with Sufu and Gli. Upon Hh signaling, Rusc is displaced from this complex, followed by dissociation of Gli from Sufu. In mammalian fibroblast cells, knockdown of Rusc2 potentiates Hh signaling by accelerating signaling-induced dissociation of the Sufu-Gli protein complexes. In Xenopus embryos, knockdown of Rusc1 or overexpression of a dominant negative Rusc enhances Hh signaling during Xenopus eye development, leading to severe eye development defects. Our study thus uncovers a novel regulatory mechanism controlling the response of cells to Hh signaling in vertebrates.

Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an antibody directed against the avain alpha 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat alpha 7 cytoplasmic domain. On sections of adult skeletal muscle the alpha 7 integrin was enriched in the myotendinous junction (MTJ). This localization was unique as neither the alpha 1, alpha 3, alpha 5, alpha 6 and alpha v subunit localizes in the myotendinous junction. The distribution of the alpha 7 subunit in the MTJ was examined during embryonic development. alpha 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the developing MTJ at this stage. alpha 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the alpha 7 subunit in the MTJ correlates with the insertion of myofibrils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the alpha 7 integrin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the alpha 7 integrin recognizes a preformed cytoskeletal structure. The presence of the alpha 7 subunit in the myotendinous junction and the alpha 5 subunit in the adhesion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Differences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.