Transgenic Soybean for Production of Thermostable α-Amylase.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes. Transient receptor potential melastatin 2 (TRPM2) activity increases in diabetic oxidative stress state, and it is involved in myocardial damage and repair. We explore the protective effect of TRPM2 knockdown on the progression of DCM. A type 2 diabetes animal model was established in C57BL/6N mice by long-term high-fat diet (HFD) feeding combined with a single injection of 100-mg/kg streptozotocin (STZ). Genetic knockdown of TRPM2 in heart was accomplished by the intravenous injection via the tail vein of adeno-associated virus type 9 carrying TRPM2 shRNA. Neonatal rat ventricular myocytes was exposed to 45 mM of high-glucose (HG) stimulation for 72 h in vitro to mimic the in vivo conditions. Western blot, real-time quantitative PCR (RT-qPCR), immunohistochemistry and fluorescence, electron, CCK-8, and flow cytometry were used to evaluate the phenotype of cardiac inflammation, fibrosis, apoptosis, and autophagy. Mice with HFD/STZ-induced diabetes exhibited systolic and diastolic dysfunction, as demonstrated by increased myocardial apoptosis and autophagy inhibition in the heart. Compared to control group, the protein expression of TRPM2, bax, cleaved caspase-3, and P62 was significantly elevated, and the protein expression of bcl-2 and LC3-II was significantly decreased in the myocardial tissues of the HFD/STZ-induced diabetes group. Knockdown of TRPM2 significantly reversed the HFD/STZ-induced myocardial apoptosis and autophagy inhibition. TRPM2 silencing attenuated HG-induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the MEK/ERK mTORC1 signaling pathway. TRPM2 knockdown attenuates hyperglycemia-induced myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice or HG-stimulated cardiomyocytes via regulating the MEK/ERK and mTORC1 signaling pathway.

Physicochemical, rheological, antioxidant and immunological properties of four novel non-inulin (poly)saccharides from Asparagus cochinchinensis.

The impacts of four extraction techniques, including hot water, ultrasonic-assisted, complex enzyme-assisted and acid-assisted methods, on the morphological, physicochemical properties and bioactivities of Asparagus cochinchinensis (poly)saccharides (EACP, WACP, UACP, and AACP) were investigated and compared. The four samples were mainly composed of glucose, fructose, and galactose with molar ratios of 50.8:22.7:4.4 for WACP, 53.9:26.0:5.3 for UACP, 35.6:14.1:21.4 for AACP and 45.0:15.6:9.0 for EACP, respectively. The rheological result showed that ACPs were non-Newtonian fluids. EACP with high purity (97.65 %) had good DPPH, O2- and ABTS+ radical scavenging activities, and significantly promoted the proliferation of the RAW264.7 cells at low concentration. UACP had good Fe2+ chelating ability, radical (DPPH, O2- and OH) scavenging activities, which might be attributed to the existence of triple-helix structure. AACP had high yield, molecular weight (17,477.2 Da), high crystallinity (23.33 %), and good radical (OH and ABTS+) scavenging activities. All four significantly stimulated the transcript expression levels of TNF-α, IL-1ß and IL-6, as determined by RT-PCR. These results suggest that the exploitation and utilization of non-inulin (poly)saccharides extracted by ultrasonic-assisted, complex enzyme-assisted and acid-assisted extraction methods are potentially valuable as effective and natural immune adjuvants and antioxidants.

Semi-Transparent, Pixel-Free Upconversion Goggles with Dual Audio-Visual Communication.

The intractable brittleness and opacity of the crystalline semiconductor restrict the prospect of developing low-cost imaging systems. Here, infrared visualization technologies are established with large-area, semi-transparent organic upconversion devices that bring high-resolution invisible images into sight without photolithography. To exploit all photoinduced charge carriers, a monolithic device structure is proposed built on the infrared-selective, single-component charge generation layer of chloroaluminum phthalocyanine (ClAlPc) coupled to two visible light-emitting layers manipulated with unipolar charges. Transient pump-probe spectroscopy reveals that the ClAlPc-based device exhibits an efficient charge dissociation process under forward bias. This process is indicated by the prompt and strong features of electroabsorption screening. Furthermore, by imposing the electric field, the ultrafast excited state dynamic suggests a prolonged charge carrier lifetime from the ClAlPc, which facilitates the charge utilization for upconversion luminance. For the first time, >30% of the infrared photons are utilized without photomultiplication strategies owing to the trivial spectrum overlap between ClAlPc and the emitter. In addition, the device can broadcast the acoustic signal by synchronizing the device frequency with the light source, which enables to operate it in dual audio-visual mode. The work demonstrates the potential of upconversion devices for affordable infrared imaging in wearable electronics.

Excited-State THz Vibrations in Aggregates of PtII Complexes Contribute to the Enhancement of Near-Infrared Emission Efficiencies.

The exploration of deactivation mechanisms for near-infrared(NIR)-emissive organic molecules has been a key issue in chemistry, materials science and molecular biology. In this study, based on transient absorption spectroscopy and transient grating photoluminescence spectroscopy, we demonstrate that the aggregated PtII complex 4H (efficient NIR emitter) exhibits collective out-of-plane motions with a frequency of 32 cm-1 (0.96 THz) in the excited states. Importantly, similar THz characteristics were also observed in analogous PtII complexes with prominent NIR emission efficiency. The conservation of THz motions enables excited-state deactivation to proceed along low-frequency vibrational coordinates, contributing to the suppression of nonradiative decay and remarkable NIR emission. These novel results highlight the significance of excited-state vibrations in nonradiative processes, which serve as a benchmark for improving device performance.

IL-13 alleviates idiopathic pulmonary hypertension by inhibiting the proliferation of pulmonary artery smooth muscle cells and regulating macrophage infiltration.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (PASMC) hyperplasia. In the present study, we conducted bioinformatic analyses and cellular experiments to assess the involvement of the interleukin-13 (IL-13) in IPAH. METHODS: The differentially expressed genes (DEGs) in IPAH and DEGs in IPAH caused by IL-13 treatment were screened using the GEO database. PPI networks were used to analyze the hub genes. Hypoxia-induced PASMCs were treated with IL-13 for in vitro assays. CCK8 and EdU staining were used to observe proliferation of PASMCs, and RT-qPCR was applied to detect the expression of hub genes. The conserved binding sites of microRNAs (miRNAs) in the 3'UTR of hub genes were investigated, and the regulatory relationships of the relevant miRNAs on their targets were verified by RT-qPCR and dual-luciferase assays. The GO and KEGG analyses were performed to study the downstream pathways. The effect of hub genes on immune cell infiltration in IPAH was investigated. RESULTS: IL-13 altered gene expression in IPAH. IL-13 inhibited the proliferation and the expression of hub genes in PASMCs. The 3'UTR sites between HNRNPA2B1, HNRNPH1, SRSF1, HNRNPU and HNRNPA3 in the hub genes and candidate regulatory miRNAs were well conserved in humans. IL-13-mediated hub genes regulated multiple pathways and influenced immune cell infiltration. Hypoxia-induced PASMCs promoted the M2 polarization of macrophages, whereas IL-13-treated PASMCs skewed the macrophages toward M1 polarization. CONCLUSIONS: IL-13-mediated alterations in hub genes inhibit PASMC proliferation and promote M1 macrophage infiltration in IPAH.

Simultaneously Enhancing Exciton/Charge Transport in Organic Solar Cells by an Organoboron Additive.

Efficient exciton diffusion and charge transport play a vital role in advancing the power conversion efficiency (PCE) of organic solar cells (OSCs). Here, a facile strategy is presented to simultaneously enhance exciton/charge transport of the widely studied PM6:Y6-based OSCs by employing highly emissive trans-bis(dimesitylboron)stilbene (BBS) as a solid additive. BBS transforms the emissive sites from a more H-type aggregate into a more J-type aggregate, which benefits the resonance energy transfer for PM6 exciton diffusion and energy transfer from PM6 to Y6. Transient gated photoluminescence spectroscopy measurements indicate that addition of BBS improves the exciton diffusion coefficient of PM6 and the dissociation of PM6 excitons in the PM6:Y6:BBS film. Transient absorption spectroscopy measurements confirm faster charge generation in PM6:Y6:BBS. Moreover, BBS helps improve Y6 crystallization, and current-sensing atomic force microscopy characterization reveals an improved charge-carrier diffusion length in PM6:Y6:BBS. Owing to the enhanced exciton diffusion, exciton dissociation, charge generation, and charge transport, as well as reduced charge recombination and energy loss, a higher PCE of 17.6% with simultaneously improved open-circuit voltage, short-circuit current density, and fill factor is achieved for the PM6:Y6:BBS devices compared to the devices without BBS (16.2%).

Expression of a Cytochrome P450 Gene from Bermuda Grass Cynodon dactylon in Soybean Confers Tolerance to Multiple Herbicides.

Bermuda grass (Cynodon dactylon) is notoriously difficult to control with some commonly used herbicides. We cloned a cytochrome P450 gene from Bermuda grass, named P450-N-Z1, which was found to confer tolerance to multiple herbicides in transgenic Arabidopsis. These herbicides include: (1) acetolactate synthase (ALS) inhibitor herbicides nicosulfuron and penoxsulam; (2) p-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide mesotrione; (3) synthetic auxin herbicide dicamba; (4) photosynthesis inhibitor bentazon. We further generated transgenic soybean plants expressing P450-N-Z1, and found that these transgenic soybean plants gained robust tolerance to nicosulfuron, flazasulfuron, and 2,4-dichlorophenoxyacetic acid (2,4-D) in greenhouse assays. A field trial demonstrated that transgenic soybean is tolerant to flazasulfuron and 2,4-D at 4-fold and 2-fold the recommended rates, respectively. Furthermore, we also demonstrated that flazasulfuron and dicamba are much more rapidly degraded in vivo in the transgenic soybean than in non-transgenic soybean. Therefore, P450-N-Z1 may be utilized for engineering transgenic crops for herbicide tolerance.

Single 3.3 fs multiple plate compression light source in ultrafast transient absorption spectroscopy.

Ultrafast transient absorption spectroscopy is a powerful tool to reveal excited state dynamics in various materials. Conventionally, probe pulses are generated via bulk supercontinuum generation or (noncollinear) optical parametric amplifiers whilst pump pulses are generated separately using (noncollinear) optical parametric amplifiers. These systems are limited by either their spectral density, stability, spectral range, and/or temporal compressibility. Recently, a new intense broadband light source is being developed, the multi-plate compression, which promises to overcome these limitations. In this paper, we analyze the supercontinuum generated by a single Multiple Plate Compression system to set a benchmark for its use in the field of ultrafast pump-probe spectroscopy. We have compressed the supercontinuum to 3.3 fs using chirp mirrors alone, making it an excellent candidate for pump-probe experiments requiring high temporal resolution. Furthermore, the single light source can be used to generate both probe and pump pulses due to its high spectral density (>14.5 nJ/nm) between 490 and 890 nm. The intensity has an average shot-to-shot relative standard deviation of 4.6 % over 490 to 890 nm, calculated over 2,000 sequential shots. By using only 1,000 shot pairs, a [Formula: see text] noise level of [Formula: see text] RMS is achieved. Finally, as a proof of concept, the transient absorption spectrum of a methylammonium lead iodide perovskite film is taken, showing great signal to noise with only 1,000 shot pairs. These results show great potential for the employment of this technique in other spectroscopic techniques such as coherent multidimensional spectroscopy.

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity.

BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters. RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain. CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

Transgenic soybean expressing a thermostable phytase as substitution for feed additive phytase.

Phytase is one of the most effective feed additives to increase the availability of phosphorus and minerals by catalyzing the hydrolysis of phytic acid. A modified appA gene (mappA) was transformed into soybean (Glycine max) under the control of a seed-specific promoter from common bean (Phaselous vulgaris). The soybean recombinant phytase showed optimal activity at pH 4.5 and 70 °C. A slight increase in enzyme activity occurred when the recombinant enzyme was pre-incubated with n-hexane. In addition, the phytase activity from our transgenic soybean does not reduce even after 2 hours of extraction with n-hexane at 55~65 °C. In conclusion, the oil extraction process using n-hexane does not inactivate the phytase expressed in the mAppA transgenic soybean, and the meal derived from the transgenic soybean processing can be used as feed supplement to livestock.

Characterization of transgenic rice expressing fusion protein Cry1Ab/Vip3A for insect resistance.

Management of resistance development of insect pests is of great importance for continued utilization of Bt crop. The high-dose/refuge and pyramid (gene stacking) strategy are commonly employed to delay the evolution of insect resistance. Due to the anticipated difficulty for deployment of mandatory refuge for transgenic crops in China, where the size of farmer is quite small, stacking of genes with different modes of action is a more feasible strategy. Here we report the development of transgenic rice expressing a fusion protein of Cry1Ab and Vip3A toxin. Analysis of trypsin proteolysis suggested that the fusion protein is equivalent to the combination of Cry1Ab and Vip3A protein. The transgenic plants expressing the fusion protein were found to be highly resistant to two major rice pests, Asiatic rice borer Chilo suppressalis (Lepidoptera: Crambidae) and rice leaf folder Cnaphalocrocis medinalis (Lepidoptera: Crambidae), while their agronomic performances showed no significant difference compared to the non-transgenic recipient rice. Therefore, the transgenic rice may be utilized for rice pest control in China.

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance.

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.

Characterization of Xylanase and Cellulase Produced by a Newly Isolated Aspergillus fumigatus N2 and Its Efficient Saccharification of Barley Straw.

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9 U/mL) and CMCase (5.61 U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65 °C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65 °C. The FPase was optimally active at pH 5.0 and 60 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.

Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment.

Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co(2+), increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase.

Carbamoyl anion-initiated cascade reaction for stereoselective synthesis of substituted α-hydroxy-ß-amino amides.

A carbamoyl anion-initiated cascade reaction with acylsilanes and imines has been used to rapidly construct substituted α-hydroxy-ß-amino amides. The Brook rearrangement-mediated cascade allows the formation of two C-C bonds and one O-Si bond in a single pot. Using this approach, a range of α-aryl α-hydroxy-ß-amino amides has been synthesized in high yields with excellent diastereoselectivities.

Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene.

Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice.

Newly Isolated Penicillium ramulosum N1 Is Excellent for Producing Protease-Resistant Acidophilic Xylanase.

Penicillium ramulosum N1 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest activities of xylanases (250 U/ml) and carboxymethyl cellulose (CMCase; 6.5 U/ml) were produced when 1% barley straw was added as a carbon source. The optimum temperature and pH for xylanase activity was 55 and 3.0 °C, respectively. The xylanases exhibited strong protease resistance. CMCase revealed maximum activities at pH 3.0 and in the range of 60-70 °C. Filter paper activity was optimally active at pH 5.0 and 55 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that there are four bands of protein with xylanase activity and three bands of proteins with endoglucanase. The results revealed that P. ramulosum N1 is a promising acidophilic and protease-resistant xylanase-producing microorganism that has great potential to be used in animal feed and food industry applications.