Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl ß- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5' untranslated region (5' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

Cobalt-free layered perovskites RBaCuFeO5+δ (R = 4f lanthanide) as electrocatalysts for the oxygen evolution reaction.

Co-based perovskite oxides are intensively studied as promising catalysts for electrochemical water splitting in an alkaline environment. However, the increasing Co demand by the battery industry is pushing the search for Co-free alternatives. Here we report a systematic study of the Co-free layered perovskite family RBaCuFeO5+δ (R = 4f lanthanide), where we uncover the existence of clear correlations between electrochemical properties and several physicochemical descriptors. Using a combination of advanced neutron and X-ray synchrotron techniques with ab initio DFT calculations we demonstrate and rationalize the positive impact of a large R ionic radius in their oxygen evolution reaction (OER) activity. We also reveal that, in these materials, Fe3+ is the transition metal cation the most prone to donate electrons. We also show that similar R3+/Ba2+ ionic radii favor the incorporation and mobility of oxygen in the layered perovskite structure and increase the number of available O diffusion paths, which have an additional, positive impact on both, the electric conductivity and the OER process. An unexpected result is the observation of a clear surface reconstruction exclusively in oxygen-rich samples (δ > 0), a fact that could be related to their superior OER activity. The encouraging intrinsic OER values obtained for the most active electrocatalyst (LaBaCuFeO5.49), together with the possibility of industrially producing this material in nanocrystalline form should inspire the design of other Co-free oxide catalysts with optimal properties for electrochemical water splitting.

Engineering the reductive tricarboxylic acid pathway in Aureobasidium pullulans for enhanced biosynthesis of poly-L-malic acid.

Aureobasidium pullulans produced poly-L-malic acid (PMA) as the main metabolite in fermentation but with relatively low productivity and yield limiting its industrial application. In this study, A. pullulans ZX-10 was engineered to overexpress cytosolic malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) and PMA synthetase (PMS) using a high-copy yeast episomal plasmid with the gpdA promoter from Aspergillus nidulans. Overexpressing endogenous PMS and heterologous MDH and PYC from Aspergillus oryzae respectively increased PMA production by 19 % - 37 % (0.64 - 0.74 g/g vs. 0.54 g/g for wild type) in shake-flask fermentations, demonstrating the importance of the reductive tricarboxylic acid (rTCA) pathway in PMA biosynthesis. A. pullulans co-expressing MDH and PYC produced 96.7 g/L PMA at 0.90 g/L∙h and 0.68 g/g glucose in fed-batch fermentation, which were among the highest yield and productivity reported. The engineered A. pullulans with enhanced rTCA pathway is advantageous and promising for PMA production.

Probing the superconducting pairing of the La4Be33Pt16alloy via muon-spin spectroscopy.

We report a study of the superconducting pairing of the noncentrosymmetric La4Be33Pt16alloy using muon-spin rotation and relaxation (µSR) technique. BelowTc=2.4 K, La4Be33Pt16exhibits bulk superconductivity (SC), here characterized by heat-capacity and magnetic-susceptibility measurements. The temperature dependence of the superfluid densityρsc(T), extracted from the transverse-fieldµSR measurements, reveals a nodeless SC in La4Be33Pt16. The best fit ofρsc(T)using ans-wave model yields a magnetic penetration depthλ0=542 nm and a superconducting gapΔ0=0.37 meV at zero Kelvin. The single-gapped superconducting state is further evidenced by the temperature-dependent electronic specific heatCe(T)/Tand the linear field-dependent electronic specific-heat coefficientγH(H). The zero-fieldµSR spectra collected in the normal- and superconducting states of La4Be33Pt16are almost identical, confirming the absence of an additional field-related relaxation and, thus, of spontaneous magnetic fields belowTc. The nodeless SC combined with a preserved time-reversal symmetry in the superconducting state proves that the spin-singlet pairing is dominant in La4Be33Pt16. This material represents yet another example of a complex system showing only a conventional behavior, in spite of a noncentrosymmetric structure and a sizeable spin-orbit coupling.

Tetrandrine Represses Inflammation and Attenuates Osteoarthritis by Selective Inhibition of COX-2.

OBJECTIVE: There is a lack of effective and long-term safe drugs for the treatment of osteoarthritis (OA). Tetrandrine (Tet) has been approved and used to treat rheumatoid arthritis for several decades, but its effect on OA has not been investigated. Herein, we explored the effect of Tet on OA and its underlying mechanism. METHODS: OA was induced using destabilization of the medial meniscus (DMM) in C57BL/6J mice. The animals were randomly divided into sham, DMM, Tet, celecoxib (CXB), and indomethacin (INDO) groups. Each group was given solvent or corresponding drugs by gavage for 7 weeks after convalescence. Pathological staining, OARSI scores, micro-computed tomography and behavior tests were performed to evaluate the effects of Tet. RESULTS: Tet remarkably alleviated cartilage injury in the knee joint, limited bone remodeling in the subchondral bone, and delayed progression of OA. Tet also significantly relieved joint pain and maintained function. Further mechanistic studies revealed that Tet lowered inflammatory cytokine levels and selectively suppressed gene and protein expression of cyclooxygenase (COX)-2 but not COX-1 (P<0.01). Tet also reduced the production of prostaglandin E2 without damaging the gastric mucosa. CONCLUSION: We found that Tet could selectively inhibit COX-2 gene expression and decrease cytokine levels in mice, thus reducing inflammation and improving OA without obvious gastric adverse events. These results provide a scientific basis for the clinical application of Tet in the treatment of OA.

Estimation of soil organic matter content in Yinchuan Plain based on fractional derivative combined with spectral indices.

Soil organic matter (SOM) is a crucial indicator of soil fertility. Field hyperspectral reflectance and laboratory SOM data of soil samples from the Yinchuan Plain were used to explore the performance of models based on fractional derivative combined with different spectral indices. Following reciprocal and logarithmic transformation, the reflectance data were processed using fractional derivative from 0 to 2 orders (interval 0.20). Then, the difference index (DI), ratio index (RI), brightness index (BI), normalized difference index (NDI), renormalized difference index (RDI), and generalized difference index (GDI) were constructed. The two-dimensional correlation between the six indices and SOM content were analyzed. The optimal spectral indices were selected to establish SOM estimation models with principal component regression (PCR), partial least square regression (PLSR), back propagation neural network (BPNN), support vector machine (SVM), and geographically weighted regression (GWR). Results showed that the maximum absolute correlation coefficient (MACC) values between DI, RI, NDI, BI, GDI, RDI, and SOM contents increased firstly and then decreased, with the highest values observed at 1.0, 0.6, 1.4, and 1.6 orders. The 0.2-2.0 order RDI under fractional derivative variation could be used for subsequent model construction, in which the optimal combinations of bands for MACC values were mainly concentrated at 400-600 nm and 1300-1700 nm. Among the different models based on the single spectral index RDI, the model based on SVM achieved the highest estimation accuracy, whose modeling determination coefficient, verification determination coefficient and relative percentage difference reached 0.86, 0.87 and 2.32. Our results would provide a scientific reference for quick and accurate SOM assessment and mapping in areas with relatively low SOM content.

Stabilizing High-Frequency Magnetic Properties of Stretchable CoFeB Films by Ribbon-Patterned Periodic Wrinkles.

The intrinsic nonstretchable feature of magnetic films has significantly limited its applications on wearable high-frequency devices. Recent studies have proved that the wrinkling surface structure based on the growth on polydimethylsiloxane (PDMS) is an effective route to obtain stretchable magnetic films. However, it is still a great challenge to simultaneously achieve a desired stretchability and stretching-insensitive high-frequency properties of magnetic films. Herein, we reported a convenient method to stabilize the high-frequency properties of stretchable magnetic films by depositing magnetic ribbon-patterned films on prestrain PDMS membranes. The ribbon-patterned wrinkling CoFeB films have far fewer cracks than the continuous film, which indicates a nice strain-relief effect and thus confers the stability of high-frequency properties for the films under stretching. However, the wrinkle bifurcation and the uneven thickness at the ribbon edge could adversely affect the stability of its high-frequency properties. The 200 µm wide ribbon-patterned film shows the best stretching-insensitive behaviors and maintains a constant resonance frequency of 3.17 GHz at strain from 10% to 25%. Moreover, a good repeatability has been demonstrated by performing thousands of stretch-release cycles, which did not significantly deteriorate its performances. The ribbon-patterned wrinkling CoFeB films with excellent stretching-insensitive high-frequency properties are promising for application in flexible microwave devices.

Biohydrogen production from brown algae fermentation: Relationship between substrate reduction degree and hydrogen production.

In this study, mannitol and mannitol-rich seaweed were fermented to investigate the relationship between substrate reduction degree and hydrogen production performance. The results showed that acetate was required in mannitol fermentation with an optimum acetate/mannitol mass ratio of 1:5. Hydrogen production and yield of mannitol fermentation reached 123.76 mL and 2.12 mol/mol-mannitol, respectively, 42.02 % and 26.95 % higher than that of glucose, respectively. The acetate was fully assimilated and the butyrate selectivity reached 100 % in the effluent. Redox potential and electron distribution showed that mannitol increased the overall electron input from mannitol and acetate, leading to the increase in hydrogen and butyrate generation. Hydrogen yield reached 2.33 mol/mol-mannitol with brown algae hydrolysate, which was the highest ever reported. This study demonstrated that substrate with a higher reduction degree could yield higher hydrogen and showed the great application potential of brown algae fermentation for the co-production of hydrogen and butyrate.

Unconventional superconductivity in topological Kramers nodal-line semimetals.

Crystalline symmetry is a defining factor of the electronic band topology in solids, where many-body interactions often induce a spontaneous breaking of symmetry. Superconductors lacking an inversion center are among the best systems to study such effects or even to achieve topological superconductivity. Here, we demonstrate that TRuSi materials (with T a transition metal) belong to this class. Their bulk normal states behave as three-dimensional Kramers nodal-line semimetals, characterized by large antisymmetric spin-orbit couplings and by hourglass-like dispersions. Our muon-spin spectroscopy measurements show that certain TRuSi compounds spontaneously break the time-reversal symmetry at the superconducting transition, while unexpectedly showing a fully gapped superconductivity. Their unconventional behavior is consistent with a unitary (s + ip) pairing, reflecting a mixture of spin singlets and spin triplets. By combining an intrinsic time-reversal symmetry-breaking superconductivity with nontrivial electronic bands, TRuSi compounds provide an ideal platform for investigating the rich interplay between unconventional superconductivity and the exotic properties of Kramers nodal-line/hourglass fermions.

High-rate continuous n-butanol production by Clostridium acetobutylicum from glucose and butyric acid in a single-pass fibrous-bed bioreactor.

Biobutanol produced in acetone-butanol-ethanol (ABE) fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs, but are rarely used in industrial fermentation because of increased risks of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At a dilution rate of 1.88 h-1 , butanol was produced at 9.55 g/L, with a yield of 0.24 g/g and productivity of 16.8 g/L/h, which was the highest productivity ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over 1 month.

Antibiofilm property and multiple action of peptide PEW300 against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen, is often associated with difficulties in treating hospital-acquired infections. Biofilms formed by P. aeruginosa significantly improve its resistance to antimicrobial agents, thereby, posing a great challenge to the combat of P. aeruginosa infection. Antimicrobial peptides (AMPs) have recently emerged as promising antibiofilm agents and increasingly attracting the attention of scientists worldwide. However, current knowledge of their antibiofilm behavior is limited and their underlying mechanism remains unclear. In this study, a novel AMP, named PEW300, with three-point mutations (E9H, D17K, and T33A) from Cecropin A was used to investigate its antibiofilm property and antibiofilm pathway against P. aeruginosa. PEW300 displayed strong antibacterial and antibiofilm activity against P. aeruginosa with no significant hemolysis or cytotoxicity to mouse erythrocyte and human embryonic kidney 293 cells. Besides, the antibiofilm pathway results showed that PEW300 preferentially dispersed the mature biofilm, leading to the biofilm-encapsulated bacteria exposure and death. Meanwhile, we also found that the extracellular DNA was a critical target of PEW300 against the mature biofilm of P. aeruginosa. In addition, multiple actions of PEW300 including destroying the cell membrane integrity, inducing high levels of intracellular reactive oxygen species, and interacting with genomic DNA were adopted to exert its antibacterial activity. Moreover, PEW300 could dramatically reduce the virulence of P. aeruginosa. Taken together, PEW300 might be served as a promising antibiofilm candidate to combat P. aeruginosa biofilms.

[Distribution Characteristics of Antibiotics and Antibiotic Resistance Genes in Manure and Surrounding Soil of Poultry Farm in Ningxia].

Antibiotics and antibiotic resistance genes (ARGs) in livestock and poultry manure pose potential ecological risks. In order to understand the distribution characteristic of antibiotics and ARGs in manure and surrounding soils of poultry farms in Ningxia, the poultry manure and relative soil samples were collected from 12 layers of different poultry breeding farms. The compositions of antibiotics and ARGs in the samples were analyzed using UPLC-MS/MS and HT-qPCR. The results showed that:① tetracycline, aminoglycoside, and sulfonamide were the dominant antibiotics in poultry manure. The types and contents of antibiotics in poultry manure were different in different breeding periods. There were more types of antibiotics in the brooding period, the average content was high, and the initial stage showed the opposite trend. ② A small amount of antibiotics was detected in the surrounding soil only 20 m away from the poultry farm, and the poultry farm had little effect on the distribution of antibiotics in the surrounding soil. The content of quinolone in the soils with poultry manure application was significantly higher than that in the control and surrounding soil. ③ We detected 132-168 ARGs in poultry manure, and the number of aminoglycosides and tetracycline was higher. The relative abundance of ARGs in the rearing period was highest, and the initial stage showed the opposite trend. The total relative abundance of ARGs in the brooding period was highest, but the terminal period showed the opposite. There were 110 ARGs in poultry manure during all breeding periods. ④ There were 23-105 ARGs in the soils, and the number of aminoglycoside was highest, followed by multidrug ARGs. The poultry farm had a great effect on the number and relative abundance of ARGs in the surrounding soil. For example, the number and relative abundance of ARGs in the surrounding soil of poultry farms gradually decreased with the increase in the distance from the poultry farms. The number and relative abundance of ARGs in the soil with applied poultry manure were significantly increased; however, these values were lower than those in the soil 20 m away from the poultry farm. ⑤ ß-lactamases, aminoglycosides, and macrolide lincosamide-streptogramin B (MLSB) ARGs were all at risk of horizontal movement in manure, and chloramphenicol ARGs were at risk of horizontal movement in soil. Correlation analysis showed that the relative abundance of aminoglycoside, tetracycline, sulfonamide, ß-lactamase, and MLSB were not significantly correlated with their contents. ⑥ Different types of ARGs had related co-occurrence phenomena, such as the positive correlation between the relative abundance of ARGs in poultry manure, and aminoglycoside and ß-lactamases, MGEs, multidrugs and vancomycins. The relative abundances of ARGs in soil, aminoglycoside and tetracyclines, vancomycins, sulfonamides, and MLSBs; tetracyclines and MLSBs; etc., all showed a significant positive correlation. In short, the co-occurrence among the relative abundance of ARGs in soil was significantly stronger than that in poultry manure. These results could provide the theoretical basis for the site selection of poultry farms, the selection of antibiotic types and dosages for large-scale breeding of laying hens, and the application of poultry manure.

Efficient Tuning of the Spin-Orbit Torque via the Magnetic Phase Transition of FeRh.

The understanding and control of the spin-orbit torque (SOT) are central to antiferromagnetic spintronics. Despite the fact that a giant SOT efficiency has been achieved in numerous materials, its efficient tuning in a given material has not been established. Materials with magnetic phase transitions (MPTs) offer a new perspective, as the SOT efficiency may vary significantly for the different magnetic orderings across the transition, and the transition itself can be readily tuned by various control parameters. This work reports that the SOT efficiency of a FeRh-based perpendicular magnetized heterostructure can be significantly tuned by varying the temperature across the MPT. The SOT efficiency exhibits a temperature hysteresis associated with the first-order nature of the MPT, and its value in the ferromagnetic phase is seen to be enhanced by ∼450%, simply by a lowering of temperature to drive FeRh into the antiferromagnetic phase. Furthermore, current-induced magnetization switching can be achieved without an assistant magnetic field for both ferromagnetic and antiferromagnetic FeRh, with a low critical switching current density for the latter. These results not only directly establish FeRh as an efficient spin generator but also present a strategy to dynamically tune SOT via varying the temperature across MPTs.

Difference responses of soil fungal communities to cattle and chicken manure composting application.

AIMS: Cattle and poultry manure composting are often applied on agricultural lands but the fungal community composition before and after application in soils is still unclear. This study describes soil fungal diversity after manure applications contribute to the correct resource use of livestock and poultry manures. METHODS AND RESULTS: Fresh manure samples were obtained from 10 beef cow farms and 12 egg-laying poultry farms at five distinct phases of rearing. Surface soil samples were collected from vegetable plots within the farms after manure application at 15, 30 and 45 t hm-2 . Using high-throughput sequencing techniques, the ITS region was used to describe soil fungus populations. The fungal OTUs, Chao1 and ACE of cattle manure were relatively higher in the fattening stage (>12 months), the OTUs and ACE of chicken manure were the highest in the initial laying stage (16-24 weeks). The fungal diversity indices of vegetable soils had no linear change after cow or chicken manure application compared with the control. Ascomycota (84.7% of total sequences), Neocallimastigomycota (9.69%) and Basidiomycota (4.6%) were the dominant phyla in cattle manure. Ascomycota (88.9%) also predominated in chicken manure followed by Basidiomycota (8.9%). Following both cattle and chicken manure applications, the abundance of Ascomycota decreased, whereas Basidiomycota and Chytridiomycota increased in the soils. None of the dominant genus increased or decreased linearly with the increase of cattle and chicken composting application rate. The fungal-dominant genera of the soils with and without manure composting application were mostly affected by soil pH and EC than manure. Pearson's correlation analysis revealed that organic matter, Cu and Hg contents were strongly linked to the fungal diversity and the abundance of specific taxa in cattle manure. In chicken manure, OM, TN and Zn were major factors controlling the fungal diversity and community composition. Soil pH, EC and Cu, Zn, Cd, Hg and As content had pronounced effects on the beneficial and pathogenic genus in soil with and without manure composting. Beneficial fungal genus such as Aspergillus, Plectosphaerella, Acremonium, Meyerozyma and fungal pathogenic such as Fusarium, Cladosporium, Verticillium were sensitive to properties (EC, pH, OM) and heavy metals (Cu, Zn, Hg) contents of the environment relatively. This study can serve as an applicable contribution helping in farms management (especially to cattle and poultry breeding) and improving their resource use of livestock and poultry manures. CONCLUSIONS: Soil heterogeneity rather than manure determines fungal communities in the vegetable fields, but we can encourage the sensible use of cattle and chicken manures in agroecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will help farmers regulate the dosage of feed components which can increase the number of beneficial fungal genus or reduce the number of pathogenic fungal genus, improve their resource use of livestock and poultry manures and encourage the sensible use of cattle and chicken manures in agroecosystems.

Multiple mobile excitons manifested as sidebands in quasi-one-dimensional metallic TaSe3.

Charge neutrality and their expected itinerant nature makes excitons potential transmitters of information. However, exciton mobility remains inaccessible to traditional optical experiments that only create and detect excitons with negligible momentum. Here, using angle-resolved photoemission spectroscopy, we detect dispersing excitons in the quasi-one-dimensional metallic trichalcogenide, TaSe3. The low density of conduction electrons and the low dimensionality in TaSe3 combined with a polaronic renormalization of the conduction band and the poorly screened interaction between these polarons and photo-induced valence holes leads to various excitonic bound states that we interpret as intrachain and interchain excitons, and possibly trions. The thresholds for the formation of a photo-hole together with an exciton appear as side valence bands with dispersions nearly parallel to the main valence band, but shifted to lower excitation energies. The energy separation between side and main valence bands can be controlled by surface doping, enabling the tuning of certain exciton properties.

Recent developments on the magnetic and electrical transport properties of FeRh- and Rh-based heterostructures.

It is fascinating how the binary alloy FeRh has been the subject of a vast number of studies almost solely for a single-phase transition. This is, however, reasonable, considering how various degrees of freedom are intertwined around this phase transition. Furthermore, the tunability of this phase transition-the large response to tuning parameters, such as electric field and strain-endows FeRh huge potential in applications. Compared to the bulk counterpart, FeRh in the thin-film form is superior in many aspects: materials in thin-film form are often more technologically relevant in the first place; in addition, the substrates add extra dimensions to the tunability, especially when the substrate itself is multiferroic. Here we review recent developments on the magnetic and transport properties of heterostructures based on FeRh and its end-member Rh, with the latter providing a new route to exploiting spin-orbit interactions in functional spintronic heterostructures other than the more often employed 5dmetals. The methods utilized in the investigation of the physical properties in these systems, and the design principles employed in the engineering thereof may conceivably be extended to similar phase transitions to other magnetic materials.

Effects of sand burial depth on Xanthium spinosum seed germination and seedling growth.

In desert habitats, sand burial is an important factor affecting germination of plant seeds and seedling growth. Xanthium spinosum has strong adaptability in arid desert areas, and is a common malignant invasive plant in Xinjiang, China. The effects of different sand burial depths on seed germination, seedling emergence, growth and biomass allocation were studied to provide a scientific basis for further control of X. spinosum. Six sand burial depths (1, 2, 3, 5, 7 and 9 cm) were established to explore the response of X. spinosum seed germination and seedling growth to sand burial. The first emergence time, peak emergence time, emergence rate, seedling growth height, biomass and biomass distribution of X. spinosum seeds was significantly different at sand burial depths (P < 0.05). The X. spinosum seeds had the highest emergence rate (71.5%) at 1 cm sand burial and the maximum seedling height (7.1 cm). As sand burial depth increased, the emergence rate and seedling height gradually decreased. Emergence rate (12.25%) and seedling height (2.9 cm) were lowest at 9 cm sand burial. The root length at 9 cm depth (13.6 cm) was significantly higher than that at other sand depths (P < 0.05). The sand burial depth affected the biomass accumulation and distribution of X. spinosum. As sand burial depth increased, the root biomass and rhizome ratio increased, and the most deeply buried seedlings allocated more biomass for root growth. The optimal sand burial depth for seed germination and seedling growth of X. spinosum was 1-3 cm, and high burial depth (5-9 cm) was not conducive to the germination and growth of X. spinosum seedlings. For prevention and control of X. spinosum, we suggest deeply ploughing crops before sowing to ensure X. spinosum seeds are ploughed into a deep soil layer.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Two-color fluorescent proteins reporting survivin regulation in breast cancer cells for high throughput drug screening.

Reporter gene assay is widely used for high throughput drug screening and drug action mechanism evaluation. In this study, we developed a robust dual-fluorescent reporter assay to detect drugs repressing the transcription of survivin, a cancer biomarker from the inhibitor of apoptosis family, in breast cancer cells cultured in three-dimensional (3D) microbioreactors. Survivin is overexpressed in numerous malignancies but almost silent in normal tissue cells and is considered a lead target for cancer therapy. Breast cancer MCF-7 cells were engineered to express enhanced green fluorescent protein driven by a survivin promoter and red fluorescent protein driven by a cytomegalovirus promoter as internal control to detect changes in survivin expression in cells as affected by drugs. This 3D dual-fluorescent reporter assay was validated with YM155 and doxorubicin, which were known to downregulate survivin in cancer cells, and further evaluated with two widely used anticancer compounds, cisplatin, and epigallocatechin gallate, to evaluate their effects on survivin expression. The results showed that the 3D dual-fluorescent reporter assay was robust for high throughput screening of drugs targeting survivin in breast cancer cells.