Investigating genetic diversity and population phylogeny of five Chongqing local chicken populations autosomal using microsatellites.

The genetic diversity and population structures of five Chongqing local chicken populations were investigated using by 24 microsatellite markers. Results revealed that the mean number of alleles (NA) ranged from 7.08 (Daninghe chicken, DN) to 8.46 (Nanchuan chicken, NC). The highest observed heterozygosity (HO) and expected heterozygosity (HE) were observed in DN (HO = 0.7252; HE = 0.7409) and the lowest HO and HE were observed in XS (Xiushan native chicken [XS], HO = 0.5910 and HE = 0.6697). The inbreeding coefficient (FIS) within population ranged from 0.022 (DN) to 0.119 (XS). Among the 24 microsatellite markers, four loci (MCW0111, MCW0016, ADL0278, and MCW0104) deviated from the Hardy-Weinberg equilibrium in all the studied populations. The results of population polygenetic analysis based on Nei's genetic distance and STRUCTURE software showed that the clustering of the five populations was incomplete consistent with geographical distribution. Moreover, a large number of gene flows were widespread among different populations, suggesting that genetic material exchanges occurred due to human activities and migration which was also verified by PCoA. In summary, this study preliminarily showed that Chongqing local chicken populations had rich genetic diversity and remarkable genetic divergence, but still high risk in conversion. These findings would be useful to the management of conservation strategies and the utilization of local chicken populations in further.

Non-KREEP origin for Chang'e-5 basalts in the Procellarum KREEP Terrane.

Mare volcanics on the Moon are the key record of thermo-chemical evolution throughout most of lunar history1-3. Young mare basalts-mainly distributed in a region rich in potassium, rare-earth elements and phosphorus (KREEP) in Oceanus Procellarum, called the Procellarum KREEP Terrane (PKT)4-were thought to be formed from KREEP-rich sources at depth5-7. However, this hypothesis has not been tested with young basalts from the PKT. Here we present a petrological and geochemical study of the basalt clasts from the PKT returned by the Chang'e-5 mission8. These two-billion-year-old basalts are the youngest lunar samples reported so far9. Bulk rock compositions have moderate titanium and high iron contents  with KREEP-like rare-earth-element and high thorium concentrations. However, strontium-neodymium isotopes indicate that these basalts were derived from a non-KREEP mantle source. To produce the high abundances of rare-earth elements and thorium, low-degree partial melting and extensive fractional crystallization are required. Our results indicate that the KREEP association may not be a prerequisite for young mare volcanism. Absolving the need to invoke heat-producing elements in their source implies a more sustained cooling history of the lunar interior to generate the Moon's youngest melts.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.

Comparative Study of Three Mixing Methods in Fusion Technique for Determining Major and Minor Elements Using Wavelength Dispersive X-ray Fluorescence Spectroscopy.

Accurate analysis using a simple and rapid procedure is always the most important pursuit of analytical chemists. In this study, a new sample preparation procedure, namely the shaker cup (SH) method, was designed and compared with two sample preparation procedures, commonly used in the laboratory, from three aspects: homogeneity of the sample-flux mixture, potential for sample contamination, and sample preparation time. For the three methods, a set of 54 certified reference materials (CRMs) was used to establish the calibration curves, while another set of 19 CRMs was measured to validate the results. In the calibration procedures, the matrix effects were corrected using the theoretical alpha coefficient method combined with the experimental coefficient method. The data of the major oxides (SiO2, TiO2, Al2O3, TFe2O3, MnO, MgO, CaO, Na2O, K2O, and P2O5) and minor elements (Cr, Cu, Ba, Ni, Sr, V, Zr, and Zn) obtained by wavelength dispersive X-ray fluorescence spectroscopy (WD-XRF) were compared using two derivative equations based on the findings by Laurence Whitty-Léveillé. The results revealed that the WD-XRF measured values using the SH method best agreed with the values recommended in the literature.

Mechanism investigation of ethosomes transdermal permeation.

Ethosomes are widely used to promote transdermal permeation of both lipophilic and hydrophilic drugs, but the mechanism of interaction between the ethosomes and the skin remains unclear. In this work, it was exploded with several technologies and facilities. Firstly, physical techniques such as attenuated total reflectance fourier-transform infrared and laser confocal Raman were used and the results indicated that the phospholipids configuration of stratum corneum changes from steady state to unstable state with the treatment of ethosomes. Differential scanning calorimetry reflected the thermodynamics change in stratum corneum after treatment with ethosomes. The results revealed that the skin of Bama mini-pigs, which is similar to human skin, treated by ethosomes had a relatively low Tm and enthalpy. Scanning electron microscopy and transmission electron microscopy showed that the microstructure and ultrastructure of stratum corneum was not damaged by ethosomes treatment. Furthermore, confocal laser scanning microscopy revealed that lipid labeled ethosomes could penetrate the skin via stratum corneum mainly through intercellular route, while during the process of penetration, phospholipids were retained in the upper epidermis. Cell experiments confirmed that ethosomes were distributed mainly on the cell membrane. Further study showed that only the drug-loaded ethosomes increased the amount of permeated drug. The current study, for the first time, elucidated the mechanistic behavior of ethosomes in transdermal application from molecular configuration, thermodynamic properties, ultrastructure, fluorescent labeling and cellular study. It is anticipated that the approaches and results described in the present study will benefit for better design of drug-loaded ethosomes.

Neural Stem Cells Transfected with Reactive Oxygen Species-Responsive Polyplexes for Effective Treatment of Ischemic Stroke.

Neural stem cells (NSCs), capable of ischemia-homing, regeneration, and differentiation, exert strong therapeutic potentials in treating ischemic stroke, but the curative effect is limited in the harsh microenvironment of ischemic regions rich in reactive oxygen species (ROS). Gene transfection to make NSCs overexpress brain-derived neurotrophic factor (BDNF) can enhance their therapeutic efficacy; however, viral vectors must be used because current nonviral vectors are unable to efficiently transfect NSCs. The first polymeric vector, ROS-responsive charge-reversal poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEA), is shown here, that mediates efficient gene transfection of NSCs and greatly enhances their therapeutics in ischemic stroke treatment. The cationic B-PDEA/DNA polyplexes can effectively transfect NSCs; in the cytosol, the B-PDEA is oxidized by intracellular ROS into negatively charged polyacrylic acid, quickly releasing the BDNF plasmids for efficient transcription and secreting a high level of BDNF. After i.v. injection in ischemic stroke mice, the transfected NSCs (BDNF-NSCs) can home to ischemic regions as efficiently as the pristine NSCs but more efficiently produce BDNF, leading to significantly augmented BDNF levels, which in turn enhances the mouse survival rate to 60%, from 0% (nontreated mice) or ≈20% (NSC-treated mice), and enables more rapid and superior functional reconstruction.

Co-delivery of deferoxamine and hydroxysafflor yellow A to accelerate diabetic wound healing via enhanced angiogenesis.

Nonhealing chronic wounds on foot induced by diabetes is a complicated pathologic process. They are mainly caused by impaired neovascularization, neuropathy, and excessive inflammation. A strategy, which can accelerate the vessel network formation as well as inhibit inflammatory response at the same time, makes it possible for effective diabetic ulcers treatment. Co-delivery of multiple drugs with complementary bioactivity offers a strategy to properly treat diabetic wound. We previously demonstrated that hydroxysafflor yellow A (HSYA) could accelerate diabetic wound healing through promoting angiogenesis and reducing inflammatory response. In order to further enhance blood vessel formation, a pro-angiogenic molecular called deferoxamine (DFO) was topically co-administrated with HSYA. The in vitro results showed that the combination of DFO and HSYA exerted synergistic effect on enhancing angiogenesis by upregulation of hypoxia inducible factor-1 alpha (HIF-1α) expression. The interpenetrating polymer networks hydrogels, characterized by good breathability and water absorption, were designed for co-loading of DFO and HSYA aiming to recruit angiogenesis relative cells and upgrade wound healing in vivo. Both DFO and HSYA in hydrogel have achieved sustained release. The in vivo studies indicated that HSYA/DFO hydrogel could accelerate diabetic wound healing. With a high expression of Hif-1α which is similar to that of normal tissue. The noninvasive US/PA imaging revealed that the wound could be recovered completely with abundant blood perfusion in dermis after given HSYA/DFO hydrogel for 28 days. In conclusion, combination of pro-angiogenic small molecule DFO and HSYA in hydrogel provides a promising strategy to productively promote diabetic wound healing as well as better the repair quality.

Synthesis, photophysical properties and biological evaluation of ß-alkylaminoporphyrin for photodynamic therapy.

A series of ß-alkylaminoporphyrins conjugated with different amines at ß position (D1-D3) or with electron-donating and electron-withdrawing substituents at phenyl position (D4-D6) were synthesized. Their photophysical and photochemical properties, intracellular localization, photocytotoxicities in vitro and vivo were also investigated. All target compounds exhibited no cytotoxicities in the dark and excellent photocytotoxicities against HeLa cells. Among them, D6 showed the highest phototoxicity and the lowest dark toxicity, which was more phototoxic than Hematoporphyrin monomethyl ether (HMME). In addition, D6 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing HeLa tumor. Therefore, D6 is a powerful and promising antitumor photosensitizer for photodynamic therapy.

Effective asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate by recombinant E. coli CCZU-A13 in [Bmim]PF6-hydrolyzate media.

It was the first report that the concentrated hydrolyzates from the enzymatic hydrolysis of dilute NaOH (3wt%)-soaking rice straw at 30°C was used to form [Bmim]PF6-hydrolyzate (50:50, v/v) media for bioconverting ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] (>99% e.e.) with recombinant E. coli CCZU-A13. Compared with pure glucose, the hydrolyzates could promote both initial reaction rate and the intracellular NADH content. Furthermore, emulsifier OP-10 (20mM) was employed to improve the reductase activity. Moreover, Hp-ß-cyclodextrin (0.01mol Hp-ß-cyclodextrin/mol COBE) was also added into this bioreaction system for enhancing the biosynthesis of (R)-CHBE from COBE by E. coli CCZU-A13 whole-cells. The yield of (R)-CHBE (>99% e.e.) from 800mM COBE was obtained at 100% in the [Bmim]PF6-hydrolyzate (50:50, v/v) media by supplementation of OP-10 (20mM) and Hp-ß-CD (8mM). In conclusion, an effective strategy for the biosynthesis of (R)-CHBE was successfully demonstrated.

Effective pretreatment of sugarcane bagasse with combination pretreatment and its hydrolyzates as reaction media for the biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by whole cells of E. coli CCZU-K14.

In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Compared with glucose, arabinose and cellobiose couldn't promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of (S)-CHBE (∼500g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of ß-CD (0.4mol ß-CD/mol COBE), l-glutamine (200mM) and glycine (500mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate (S)-CHBE.

Effective pretreatment of dilute NaOH-soaked chestnut shell with glycerol-HClO4-water media: structural characterization, enzymatic saccharification, and ethanol fermentation.

In this study, an effective pretreatment of dilute NaOH-soaked chestnut shell (CNS) with glycerol-HClO4-water (88.8:1.2:10, w/w/w) media at 130 °C for 30 min was successfully demonstrated. Results revealed that the combination pretreatment removed 66.0 % of lignin and 73.7 % of hemicellulose in untreated CNS. The changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS were characterized with Fourier transform infrared spectroscopy, fluorescent microscope, scanning electron microscopy, and X-ray diffraction. Biotransformation of glycerol-HClO4-water pretreated-NaOH-soaked CNS (50 g/L) with a cocktail of enzymes for 72 h, the reducing sugars and glucose were 39.7 and 33.4 g/L, respectively. Moreover, the recovered hydrolyzates containing 20 g/L glucose had no inhibitory effects on the ethanol-fermenting microorganism, and the ethanol production was 0.45 g/g glucose within 48 h. In conclusion, this combination pretreatment shows promise as pretreatment solvent for wheat straw, although the in-depth exploration of this subject is needed.

Enzymatic in situ saccharification of chestnut shell with high ionic liquid-tolerant cellulases from Galactomyces sp. CCZU11-1 in a biocompatible ionic liquid-cellulase media.

In this study, it was the first time to report that the cellulases of Galactomyces sp. CCZU11-1 showed high activity and stability in the culture and reaction media containing IL [Mmim]DMP. Using untreated chestnut shell (CNS) as carbon source in the culture media containing IL [Mmim]DMP (5%, w/v), high activity of FPA (28.6U/mL), xylanase (186.2U/mL), and CMCase (107.3U/mL) were obtained, and 184.9mg/L of total protein was achieved. Furthermore, the changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS utilized with Galactomyces sp. CCZU11-1 were characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. After was enzymatically hydrolyzed with the prepared crude enzymes in IL diluted to 20% (w/v), a high yield of reducing sugars, 62.1%, was obtained. Significantly, Galactomyces sp. CCZU11-1 showed high potential for the efficient transformation of lignocellulosic materials to glucose in a single-step process.

Enhancement of enzymatic saccharification of corn stover with sequential Fenton pretreatment and dilute NaOH extraction.

In this study, an effective method by the sequential Fenton pretreatment and dilute NaOH extraction (FT-AE) was chosen for pretreating corn stover. Before dilute NaOH (0.75 wt%) extraction at 90 °C for 1h, Fenton reagent (0.95 g/L of FeSO4 and 29.8 g/L of H2O2) was employed to pretreat CS at a solid/liquid ratio of 1/20 (w/w) at 35 °C for 30 min. The changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated solid residue were correlated with the enhancement of enzymatic saccharification. After being enzymatically hydrolyzed for 72 h, the reducing sugars and glucose from the hydrolysis of 60 g/L FT-AE-CS pretreated could be obtained at 40.96 and 23.61 g/L, respectively. Finally, the recovered hydrolyzates containing glucose had no inhibitory effects on the ethanol fermenting microorganism. In conclusion, the sequential Fenton pretreatment and dilute NaOH extraction has high potential application in future.

Significantly improving enzymatic saccharification of high crystallinity index's corn stover by combining ionic liquid [Bmim]Cl-HCl-water media with dilute NaOH pretreatment.

In this study, a pretreatment by combining acidified aqueous ionic liquid 1-butyl-3-methylimidazolium chloride (IL [Bmim]Cl) solution with dilute NaOH extraction was employed to pretreat high crystallinity index (CrI) of corn stover before its enzymatic saccharification. After NaOH extraction, [Bmim]Cl-HCl-water (78.8:1.2:20, w/w/w) media was used for further pretreatment at 130 °C for 30 min. After being enzymatically hydrolyzed for 48 h, corn stover pretreated could be biotransformed into reducing sugars in the yield of 95.1%. Furthermore, SEM, XRD and FTIR analyses of untreated and pretreated corn stovers were examined. It was found that the intact structure was disrupted by combination pretreatment and resulted in a porous and amorphous regenerated cellulosic material that greatly improved enzymatic hydrolysis. Finally, the recovered hydrolyzates obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. In conclusion, the combination pretreatment shows high potential application in future.

Effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate by supplementation of l-glutamine, d-xylose and ß-cyclodextrin in n-butyl acetate-water media.

To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-glutamine (200mM) and d-xylose (250mM) was added into in n-butyl acetate-water (10:90, v/v) biphasic system instead of NAD(+) for increasing the biocatalytic efficiency. To further improve the synthesis of optically pure ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee), ß-cyclodextrin was also added into this reaction media, and ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee) could be effectively synthesized from 800mM ethyl 4-chloro-3-oxobutanoate in the yield of 100% by whole-cells of recombinant E. coli CCZU-A13. Finally, the possible mechanism for improving the reductase activity by supplementation of l-glutamine, d-xylose and ß-CD was proposed. In conclusion, this strategy has high potential for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee).

Improved biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by adding L-glutamine plus glycine instead of NAD+ in ß-cyclodextrin-water system.

To reduce dependence on the expensive cofactor and effectively biotransform ethyl 4-chloro-3-oxobutanoate, L-glutamine and glycine were found to enhance the content of intracellular NADH and the reductase activity. Adding the mixture of 200 mM of L-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of reductase activity was increased over the control without the addition of the two compounds. Moreover, ß-cyclodextrin (0.4 mol ß-cyclodextrin/mol ethyl 4-chloro-3-oxobutanoate) was also added into this reaction media, and the biocatalytic activity of the whole-cell biocatalyst of Escherichia coli CCZU-K14 was increased by 1.34-fold than that without ß-cyclodextrin. In this ß-cyclodextrin-water media containing L-glutamine (200 mM) plus glycine (500 mM), ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained from 3000 mM ethyl 4-chloro-3-oxobutanoate in the yield of 98.0% after 8h. All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of ethyl (S)-4-chloro-3-hydroxybutanoate.

Biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with an NADH-dependent reductase (ClCR) discovered by genome data mining using a modified colorimetric screening strategy.

An NADH-dependent reductase (ClCR) was discovered by genome data mining. After ClCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-T15 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was screened using a modified high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (S)-CHBE (>99% ee) with the resting cells of E. coli CCZU-T15 was successfully demonstrated in toluene-water (50:50, v/v) biphasic system. Biotransformation of 1000 mM COBE for 24 h in the biphasic system, (S)-CHBE (>99% ee) could be obtained in the high yield of 96.4%. Significantly, E. coli CCZU-T15 shows high potential in the industrial production of (S)-CHBE (>99% ee).

Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by Rhodococcus sp.

Rhodococcus sp. CGMCC 4911 transformed 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives into corresponding diols. Ethylene sulfate, glycol sulfide, 1,3-PDS, and 1,2-propanediol cyclic sulfate were effectively hydrolyzed with growing cells. (R)-1,2-Propanediol (>99 % e.e.) was obtained at 44 % yield with growing cells. Glycol sulfide, ethylene sulfate, and 1,3-PDS were converted into the corresponding diols at 94.6, 96.3, and 98.3 %, respectively. Optimal reaction conditions with lyophilized resting cells were 30 °C, pH 7.5, and cell dosage 17.9 mg cell dry wt/ml. 1,3-Propanediol was obtained from 50 mM 1,3-PDS at 97.2 % yield by lyophilized cells after 16 h. Lyophilized cells were entrapped in calcium alginate with a half-life of 263 h at 30 °C, and the total operational time of the immobilized biocatalysts could reach over 192 h with a high conversion rate.

Improving enzymatic hydrolysis of corn stover pretreated by ethylene glycol-perchloric acid-water mixture.

To improve the enzymatic saccharification of lignocellulosic biomass, a mixture of ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) was used for pretreating corn stover in this study. After the optimization in oil-bath system, the optimum pretreatment temperature and time were 130 °C and 30 min, respectively. After the saccharification of 10 g/L pretreated corn stover for 48 h, the saccharification rate was obtained in the yield of 77.4 %. To decrease pretreatment temperature and shorten pretreatment time, ethylene glycol-HClO4-water (88.8:1.2:10, w/w/w) media under microwave irradiation was employed to pretreat corn stover effectively at 100 °C and 200 W for 5 min. Finally, the recovered hydrolyzates containing glucose obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. These results would be helpful for developing a cost-effective pretreatment combined with enzymatic saccharification of cellulosic materials for the production of lignocellulosic ethanol.