An Efficient Deacidification Process for Safflower Seed Oil with High Nutritional Property through Optimized Ultrasonic-Assisted Technology.

Safflower seed oil (SSO) is considered to be an excellent edible oil since it contains abundant essential unsaturated fatty acids and lipid concomitants. However, the traditional alkali-refined deacidification process of SSO results in a serious loss of bioactive components of the oil and also yields massive amounts of wastewater. In this study, SSO was first extracted by ultrasonic-assisted ethanol extraction (UAEE), and the extraction process was optimized using random centroid optimization. By exploring the effects of ethanol concentration, solid−liquid ratio, ultrasonic time, and the number of deacidification times, the optimum conditions for the deacidification of safflower seed oil were obtained as follows: ethanol concentration 100%, solid−liquid ratio 1:4, ultrasonic time 29 min, and number of deacidification cycles (×2). The deacidification rate was 97.13% ± 0.70%, better than alkali-refining (72.16% ± 0.13%). The values of acid, peroxide, anisidine and total oxidation of UAEE-deacidified SSO were significantly lower than those of alkali-deacidified SSO (p < 0.05). The contents of the main lipid concomitants such as tocopherols, polyphenols, and phytosterols in UAEE-decidified SSO were significantly higher than those of the latter (p < 0.05). For instance, the DPPH radical scavenging capacity of UAEE-processed SSO was significantly higher than that of alkali refining (p < 0.05). The Pearson bivariate correlation analysis before and after the deacidification process demonstrated that the three main lipid concomitants in SSO were negatively correlated with the index of peroxide, anisidine, and total oxidation values. The purpose of this study was to provide an alternative method for the deacidification of SSO that can effectively remove free fatty acids while maintaining the nutritional characteristics, physicochemical properties, and antioxidant capacity of SSO.

Hg2+ detection, pH sensing and cell imaging based on bright blue-fluorescent N-doped carbon dots.

A multifunctional sensing platform based on bright blue-fluorescent nitrogen-doped carbon dots (N-CDs) has been ingeniously designed for the sensitive determination of Hg2+ and pH. The N-CDs were facilely fabricated via a one-step hydrothermal treatment of citric acid and folic acid with admirable merits including exceptional stability, low toxicity and distinguished biocompatibility. Intriguingly, the obtained N-CDs can be utilized to detect Hg2+ on the basis of the dynamic quenching effect, showing a linear range of 0 µM-400 µM as well as a detection limit of 0.124 µM. In addition, the obtained N-CDs revealed a significant emission enhancement with increasing pH from 4.0 to 10.0 and acquired a good linearity in the pH range of 6.8-7.8. Taking advantage of efficient analysis of Hg2+ and pH in aqueous solution, the as-synthesized N-CDs have been extended to the visualization biosensing platform to timely track Hg2+ and pH in living cells.

Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.

BACKGROUND: Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. RESULTS: We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. CONCLUSIONS: We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

Transition Metal Phosphide Nanoparticles Supported on SBA-15 as Highly Selective Hydrodeoxygenation Catalysts for the Production of Advanced Biofuels.

A series of catalysts constituted by nanoparticles of transition metal (M = Fe, Co, Ni and Mo) phosphides (TMP) dispersed on SBA-15 were synthesized by reduction of the corresponding metal phosphate precursors previously impregnated on the mesostructured support. All the samples contained a metal-loading of 20 wt% and with an initial M/P mole ratio of 1, and they were characterized by X-ray diffraction (XRD), N2 sorption, H2-TPR and transmission electron microscopy (TEM). Metal phosphide nanocatalysts were tested in a high pressure continuous flow reactor for the hydrodeoxygenation (HDO) of a methyl ester blend containing methyl oleate (C17H33-COO-CH3) as main component (70%). This mixture constitutes a convenient surrogate of triglycerides present in vegetable oils, and following catalytic hydrotreating yields mainly n-alkanes. The results of the catalytic assays indicate that Ni2P/SBA-15 catalyst presents the highest ester conversion, whereas the transformation rate is about 20% lower for MoP/SBA-15. In contrast, catalysts based on Fe and Co phosphides show a rather limited activity. Hydrocarbon distribution in the liquid product suggests that both hydrodeoxygenation and decarboxylation/decarbonylation reactions occur simultaneously over the different catalysts, although MoP/SBA-15 possess a selectivity towards hydrodeoxygenation exceeding 90%. Accordingly, the catalyst based on MoP affords the highest yield of n-octadecane, which is the preferred product in terms of carbon atom economy. Subsequently, in order to conjugate the advantages of both Ni and Mo phosphides, a series of catalysts containing variable proportions of both metals were prepared. The obtained results reveal that the mixed phosphides catalysts present a catalytic behavior intermediate between those of the monometallic phosphides. Accordingly, only marginal enhancement of the yield of n-octadecane is obtained for the catalysts with a Mo/Ni ratio of 3. Nevertheless, owing to this high selectivity for hydrodeoxygenation MoP/SBA-15 appears as a very promising catalyst for the production of advanced biofuels.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.

Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

Long wavelength emission fluorescent probe for highly selective detection of cysteine in living cells.

We developed a fluorescent probe, named 2-(4-(acryloyloxy) phenyl)-4-(2-carboxyphenyl)-7-(diethylamino) chromenylium (PA-A), for detecting Cys using the -OH protection/deprotection strategy, which can react with Cys to form a red-emitting anthocyanidin derivative fluorophore. The probe has high selectivity to Cys over Hcy and GSH in phosphate buffer solution (PBS, 10 mM, pH = 7.4), high sensitivity, a low detection limit of 4.48 × 10-8 mol/L, and it can be recognized with the naked eye. Fluorescence imaging experiment of Cys with PA-A at the cellular successfully showed excellent tissue penetration.

A design optimized prime editor with expanded scope and capability in plants.

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in plant breeding. Prime editing, a versatile gene-editing approach that directly writes new genetic information into a specified DNA site without requiring double-strand DNA breaks, suffers from low efficiency in plants1-5. In this study, N-terminal reverse transcriptase-Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion. In addition, introduction of multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced efficiency. By using these two methods synergistically, prime editing with an average editing frequency as high as 24.3% at 13 endogenous targets in rice transgenic plants, 6.2% at four targets in maize protoplasts and 12.5% in human cells was achieved, which is two- to threefold higher than the original editor, Prime Editor 3. Therefore, our optimized approach has potential to make more formerly non-editable target sites editable, and expands the scope and capabilities of prime editing in the future.

Iron ion sensing and in vitro and in vivo imaging based on bright blue-fluorescent carbon dots.

Herein, we propose an eco-friendly synthesis of carbon dots (CDs) and ingeniously design a rapid and label-free "turn-off" sensing platform for ultrasensitive recognition of Fe3+ in vitro and in vivo. CDs with extraordinary advantages involving exceptional stability, ultra-low toxicity as well as admirable biocompatibility were simply prepared via one-step hydrothermal strategy of Caulis polygoni multiflora. Result indicated that as-acquired CDs not only exhibit excitation dependency, but also have a high quantum yield of (QY) up to 42%. Miraculously, the fluorescence of CDs can be extinguished sharply by Fe3+ because of static quenching effect with linear range of 0-400 µM, yielding a detection limit of 0.025 µM. Benefiting from these characteristics, CDs have been extended for multicolourful imaging and tracking Fe3+ fluctuations in living cells. Bioimaging of zebrafish larvae exposed to CDs confirmed that it is smoothly circulated to other tissues and organs owing to their small size. Eventually, as-prepared CDs have been implemented for the real-time detection of Fe3+ in nude mice.

Lysosome-targeted carbon dots for colorimetric and fluorescent dual mode detection of iron ion, in vitro and in vivo imaging.

In this work, a colorimetric and fluorescent dual mode sensor based on lysosome-targeted CDs has been desirably implemented to identify Fe3+ fluctuations in vitro and in vivo. By simple one-pot hydrothermal carbonization of dried field mint, yellow-fluorescent CDs were directly fabricated without the assistance of other reagents and hold exceptional stability, superior biocompatibility as well as ultra-low cytotoxicity. Results indicated that as-prepared CDs can provide a rapid, reliable, and highly selective recognition of Fe3+ with a linear range of 0 µM-400 µM and a detection limit of 0.037 µM. Impressively, it was found that as-developed CDs can successfully target lysosome with high colocalization coefficient (0.85) and responds to fluctuations of Fe3+ in living cells. Further, acquired CDs was ingeniously devoted to Escherichia coli imaging. Besides, obtained CDs was eventually utilized to track the variation of Fe3+ in vivo system. A preliminary research expresses that as-synthesized CDs can function as an effective tool to detect Fe3+ in vitro and in vivo and thus indicates the promising applicability for disease detection in physiology and pathology in the future.

The effect of hydrothermal temperature on the properties of SBA-15 materials.

In present work, ordered mesoporous material SBA-15 was synthesized by using poly (alkylene oxide) block copolymer (Pluronic P123) as template and ethylsilicate as silica source in weak acid environment in a wide range of temperature. The focus of synthesis research was high hydrothermal temperature. The obtained products were characterized by various techniques, including XRD, N2 sorption isotherms, FTIR spectroscopy and thermogravimetric. The effect of hydrothermal temperature on the specific surface area, pore volume and pore size of SBA-15 products was investigated systematically. As the hydrothermal temperature increases from the 100-120 °C, the specific surface area and the pore volume of the mesoporous molecular sieve increase greatly. When the hydrothermal temperature increase further, the pore volume of the mesoporous molecular sieve increase continually. But the specific surface area decrease significantly. When the hydrothermal temperature is too high (over 140 °C), the order degree begins to decrease, So the specific surface area and pore volume decrease significantly because the pores structure have significant destruction and collapse. Mechanism and structural characteristics of P123 block copolymer could explain in detail the effect of hydrothermal temperature on the property and structure of mesoporous molecular sieve SBA-15.

A resazurin-based, nondestructive assay for monitoring cell proliferation during a scaffold-based 3D culture process.

Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering. Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation. The present work found that cytotoxicity and depletion of resazurin depended on cell concentration, resazurin concentration and resazurin incubation time. A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin. This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(d,l-lactic acid) scaffold during a continuous 3D culture process for up to 21 days, comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability, proliferation and differentiation functions of MC3T3-E1, which were treated with/without this strategy for nondestructive evaluation. This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay. No major negative effects of this method on MC3T3-E1 viability and functions were found. Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.

Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility.

The CRISPR-Cas9 system enables simple, rapid, and effective genome editing in many species. Nevertheless, the requirement of an NGG protospacer adjacent motif (PAM) for the widely used canonical Streptococcus pyogenes Cas9 (SpCas9) limits the potential target sites. The xCas9, an engineered SpCas9 variant, was developed to broaden the PAM compatibility to NG, GAA, and GAT PAMs in human cells. However, no knockout rice plants were generated for GAA PAM sites, and only one edited target with a GAT PAM was reported. In this study, we used tRNA and enhanced sgRNA (esgRNA) to develop an efficient CRISPR-xCas9 genome editing system able to mutate genes at NG, GAA, GAT, and even GAG PAM sites in rice. We also developed the corresponding xCas9-based cytosine base editor (CBE) that can edit the NG and GA PAM sites. These new editing tools will be useful for future rice research or breeding, and may also be applicable for other related plant species.

Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells.

The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.

Bone marrow derived endothelial progenitor cells retain their phenotype and functions after a limited number of culture passages and cryopreservation.

A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and post-cryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications.

Oxidative desulfurization of dibenzothiophene with molecular oxygen using emulsion catalysis.

Dibenzothiophene (DBT) is oxidized to the corresponding sulfoxide and sulfone in an emulsion system (W/O) composed of polyoxometalate anion [C(18)H(37)N(CH(3))3](5)[PV(2)Mo(10)O(40)] as both the surfactant and catalyst, using molecular oxygen as the oxidant and aldehyde as the sacrificial agent under mild conditions.

The study of thiophene adsorption onto La(III)-exchanged zeolite NaY by FT-IR spectroscopy.

Zeolites NaY and LaNaY (ion-exchanged with aqueous lanthanum nitrate solution) were used as adsorbents for removing organic sulfur compounds from model gasoline solutions (without and with toluene) and fluid catalytic cracked gasoline in fixed-bed adsorption equipment at room temperature and atmosphere pressure. The adsorptive selectivity for organic sulfur compounds was significantly increased when Na(+) ions in zeolite NaY were exchanged with lanthanum ions. IR spectra of thiophene adsorption indicate that thiophene is adsorbed onto La(3+) ions via direct S-La(3+) interaction and Na(+) ions via pi-electronic interaction for La(3+)-exchanged zeolite NaY, but only via pi-electronic interaction with Na(+) ions for NaY. The amount of adsorbed thiophene on La(3+)-exchanged zeolite Y was slightly decreased by coadsorption of benzene, but greatly reduced on NaY. The adsorption of thiophene via interaction with La(3+) on La(3+)-exchanged zeolite Y is hardly replaced by benzene coadsorption. The direct S-La(3+) interaction might be the essential reason for the evidently improved adsorptive selectivity of LaNaY for removing organic sulfur compounds from solutions containing large amount of aromatics.

[Changes in vegetation and soil characteristics under tourism disturbance in lakeside wetland of northwest Yunnan Plateau, Southwest China].

The characteristics of vegetation and soil were investigated in Bita Lake and Shudu Lake wetlands in northwest Yunnan Plateau under tourism disturbance. The 22 typical plots in the wetlands were classified into 4 types by TWINSPAN, including primary wetland, light degradation, moderate degradation, and severe degradation. Along the degradation gradient, the plant community density, coverage, species number and Shannon diversity index increased and the plant height decreased in Bita Lake and Shudu Lake wetlands, and Whittaker diversity index increased in Bita Lake wetland. Plant species number, soil organic matter, total nitrogen, porosity, available nitrogen, available phosphorus and available potassium contents were higher in Shudu Lake wetland than in Bita Lake wetland, but the plant density, height, soil total potassium and pH were opposite. Canonical correspondence analysis (CCA) by importance values of 42 plants and 11 soil variables showed that soil organic matter, total nitrogen and total potassium were the key factors on plant species distribution in Bita Lake and Shudu Lake wetlands under tourism disturbance. TWINSPAN classification and analysis of vegetation-soil characteristics indicated the effects of tourism disturbance in Bita Lake wetland were larger than in Shudu Lake wetland.

[Evaluation index system of swamp degradation in Zoige Plateau of Sichuan, Southwest China under drainage stress].

The evaluation index system of swamp degradation is one of the key scientific issues in the frontier field of international wetland science research. On the basis of long-term swamp field reconnaissance, and according to the fixed position ecological investigation of plant communities and the analysis of soil samples in 20 swamp plots in three belt transects of swamp degradation research under the stress of drainage in 2009, the swamps in the Zoige Plateau of Sichuan were classified into three groups with seven swamp communities, i. e., undisturbed (A type), disturbed by long-term and weak drainage (B-D type), and disturbed by short-term and strong drainage (E-G type), according to the species importance value and by Two-Way Indicator Species Analysis (TWINSPAN). The degradation degree of the swamps was graded by the method of Principal Components Analysis (PCA), and the swamp vegetation evaluation index (SVEI) and soil evaluation index (SSEI) were developed. Based on the SVEI, the swamps were classified as pristine swamp, lightly degraded swamp, moderately degraded swamp, and severely degraded swamp. Based on the SSEI, the swamps in Hongyuan County were divided into three grades, i. e. pristine swamp, lightly degraded swamp, and severely degraded swamp, while those in Ruoergai County were divided into lightly degraded swamp, moderately degraded swamp, and severely degraded swamp. The similarity of TWINSPAN classification results and SVEI/SSEI evaluation results was above 70%, indicating that both SVEI and SSEI were effective for the swamp degradation grading, and different classification methods should be combined to comprehensively evaluate the swamps in the Plateau.

[Degradation characteristics, patterns, and processes of lakeside wetland in Napahai of northwest Yunnan Plateau, Southwest China].

Two-way indicator species analysis (TWINSPAN) and canonical correspondence analysis (CCA) were employed to analyze the degradation characteristics, patterns, and processes of lakeside wetland in Napahai of northwest Yunnan Plateau. The plant communities in the lakeside wetland could be classified into four associations, according to the TWINSPAN results. The succession patterns of the plant community were represented by aquatic plant community --> swamp plant community --> swamp meadow plant community --> meadow plant community. During the processes of succession, the plant community coverage, density, Shannon diversity index, species richness, and plant aboveground biomass increased, whereas the community height decreased. The succession of the plant hydro-ecotypes followed the sequence of aquatic --> helophyte --> hygrophyte --> mesophyte. With the succession of the plant community, the wetland water quality indices salinity, alkalinity, and hardness decreased but ammonium nitrogen and total phosphorus concentrations increased, while total nitrogen and nitrate nitrogen concentrations showed no significant changes. Meanwhile, the soil pH and soil organic matter and total nitrogen contents decreased gradually, soil total phosphorous and potassium contents were increasing, whereas soil available phosphorous and potassium contents decreased after an initial increase. The CCA showed that the community species composition and structure were mainly affected by the wetland water gradient. The soil pH and total phosphorus content and the water total nitrogen and ammonium nitrogen concentrations had significant effects on the wetland plant species distribution and plant community succession.