An optimization by response surface methodology for the enhanced production of rMBSP from Pichia pastoris and study of its application.

Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties.Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter.Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca2+, Mg2+, SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n-hexane and n-heptane. Enzymatic analysis revealed a Km of 2.89 ± 0.09 µM and a Vmax of 14.20 ± 0.12 nM•min-1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP.Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research.

Effects of different light conditions on transient expression and biomass in Nicotiana benthamiana leaves.

In the process of the production of recombinant proteins by using an Agrobacterium-mediated transient gene expression system, the effectiveness of the control of light conditions pre- and post-agroinfiltration on efficiency of transient expression is worth being evaluated. In this study, Nicotiana benthamiana plants were used as a bioreactor to investigate the effects of different light conditions pre- and post-agroinfiltration on the transient expression of green fluorescent protein (GFP). The results showed that the plants grown under light condition for 5 weeks had the highest level of transient expression among those grown for 4-8 weeks. In the pre-agroinfiltration, the level of transient expression of GFP was obviously decreased by the increase in light intensity or by the shortening of the photoperiod. Although the shortening of the photoperiod post-agroinfiltration also decreased the level of transient expression, moderate light intensity post-agroinfiltration was needed for higher level of transient expression efficiency. However, there was no strong correlation between the transient expression efficiency and plant growth. The results suggested that light condition was an important factor affecting the level of transient expression in plants. Hence, light conditions should be optimized to obtain higher productivity of recombinant protein from transient expression systems.

[Expression of antigens of foot-and-mouth disease virus in plants: a review].

Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV), which seriously endangers the development of animal husbandry. The inactivated FMD vaccine is the main product for the prevention and control of FMD, which has been successfully applied to control the pandemic and outbreak of FMD. However, the inactivated FMD vaccine also has problems, such as the instability of antigen, the risk of spread of the virus due to incomplete inactivation during vaccine production, and the high cost of production. Compared with traditional microbial and animal bioreactors, production of antigens in plants through transgenic technology has some advantages including low cost, safety, convenience, and easy storage and transportation. Moreover, since antigens produced from plants can be directly used as edible vaccines, no complex processes of protein extraction and purification are required. But, there are some problems for the production of antigens in plants, which include low expression level and poor controllability. Thus, expressing the antigens of FMDV in plants may be an alternative mean for production of FMD vaccine, which has certain advantages but still need to be continuously optimized. Here we review the main strategies for expressing active proteins in plants, as well as the research progress on the expression of FMDV antigens in plants. We also discuss the current problems and challenges encountered, with the aim to facilitate related research.

Development of three-dimensionally printed vascular stents of bioresorbable poly(l-lactide-co-caprolactone).

With the ripening of 3D printing technology and the discovery of a variety of printable materials, 3D-printed vascular stents provide new treatment options for patients with angiocardiopathy. Bioresorbable stent not only combines the advantages of metallic stent and drug-coated balloon, but also avoids the disadvantages of them. 3D printing is also an economical and efficient way to produce stents and makes it possible to construct complex structures. In this study, stents made from poly(l-lactic acid) (PLLA), poly(ε-caprolactone) (PCL) and poly(l-lactide-co-caprolactone) (PLCL) were manufactured by 3D printing and evaluated for radial strength, crystallinity and molecular weight. PLCL copolymerized by different proportions of lactic acid and caprolactone showed different mechanical and degradation properties. This demonstrated the potential of 3D printing as a low-cost and high throughput method for stent manufacturing. The PLLA and PLCL 95/5 stents had similar mechanical properties, whereas PLCL 85/15 and PCL stents both had relatively low radial strength. In general, PLCL 95/5 had a faster degradation rate than PLLA. These two materials were made into peripheral vascular bioresorbable scaffolds (BRS) and further studied by additional bench testing. PLCL 95/5 peripheral BRS had superior mechanical properties in terms of flexural/bending fatigue and compression resistance.

Alleviative effects of magnetic Fe3O4 nanoparticles on the physiological toxicity of 3-nitrophenol to rice (Oryza sativa L.) seedlings.

In the present study, we explored whether magnetic iron oxide nanoparticles (MNPs-Fe3O4) can be used to alleviate the toxicity of 3-nitrophenol (3-NP) to rice (Oryza sativa L.) seedlings grown under hydroponic conditions. The results showed that 3-NP from 7 to 560 µM decreased the growth, photochemical activity of the photosystem II (PS II), and chlorophyll content of the seedlings in a concentration-dependent manner. In the presence of 3-NP, 2,000 mg L-1 MNPs-Fe3O4 were added to the growth medium as the absorbents of 3-NP and then were separated with a magnet. The emergence of MNPs-Fe3O4 effectively alleviated the negative effects of 3-NP on rice seedlings. In addition, the long-term presence of MNPs-Fe3O4 (from 100 to 2,000 mg L-1) in the growth medium enhanced the growth, production of reactive oxygen species (ROS), activities of antioxidant enzymes, photochemical activity of PS II, and chlorophyll content of the rice seedlings. These results suggest that MNPs-Fe3O4 could be used as potential additives to relieve the physiological toxicity of 3-NP to rice seedlings.

The involvement of extracellular ATP in regulating the stunted growth of Arabidopsis plants by repeated wounding.

BACKGROUND: Extracellular ATP (exATP) has been shown to act as a signal molecule for regulating growth, development, and responses of plants to the external environment. RESULTS: In this study, we investigated the possible involvement of exATP in regulating the stunted growth caused by repeated wounding. The present work showed that the repeated wounding caused the decreases in leaf area, fresh weight, dry weight, and root length of Arabidopsis seedlings, while the exATP level was enhanced by the repeated wounding. Repeated application of exogenous ATP had similar effects on the plant growth, as the repeated wounding. Through the comparison of p2k1-3 mutant (in which T-DNA disrupted the gene coding P2K1, as exATP receptor) and wide type (WT) plants, it was found that the mutation in P2K1 decreased the sensitivity of plant growth to the repeated wounding and exogenous ATP application. Further works showed that the ibuprofen (IBU, an inhibitor of jasmonate biosynthesis) partially rescued the wound-induced growth degradation. In comparison, the P2K1 mutation partly rescued the wound-induced growth degradation, whereas this mutation failed to do so in the wounded seedlings treated with IBU, indicating that the role of exATP in regulating the growth degradation by repeated wounding could be linked to the JA signaling pathway. CONCLUSIONS: In conclusion, these results indicate that exATP could be a regulator for the stunted growth of plants by repeated wounding.

Extracellular ATP is involved in regulating Arabidopsis seed germination.

MAIN CONCLUSION: Extracellular ATP level induced a transient increase during germination of Arabidopsis seeds, and extracellular ATP could negatively regulate the seed germination by its receptor, DORN1. Extracellular ATP (exATP) acts as a signal molecule for regulating growth, development, and responses of plants to external environments. In this study, we investigated the possible involvement of exATP in regulating the seed germination of Arabidopsis thaliana. Treatments of Arabidopsis seeds with exogenous ATP delayed seed germination, suggesting that exATP could be a repressor for seed germination. During the germination of Arabidopsis seeds, the exATP level of the seeds presented a transient increase. When exogenous application of the glucose-hexokinase system effectively decreased the exATP level of the Arabidopsis seeds during germination, the percentage of germination was significantly enhanced, while the products of ATP hydrolysis had no effects on the germination. Further studies showed that the seeds of dorn 1-3 mutant plants (mutation in exATP receptor) showed a higher germination percentage, compared to the seeds of wide type (WT) plants. In addition, the dorn 1-3 mutant seeds were less sensitive to the delay-effect of exogenous ATP on seed germination than the WT seeds. The dorn 1-3 mutant seeds presented a higher GA (gibberellin) content, lower ABA (abscisic acid) content, and lower ratio of ABA/GA contents before the imbibition, compared to the WT seeds. The regulation of seed germination by exATP was dependent on the external temperature. These data suggest that exATP is involved in regulating Arabidopsis seed germination.

Two-stage degradation and novel functional endothelium characteristics of a 3-D printed bioresorbable scaffold.

Bioresorbable scaffolds have emerged as a new generation of vascular implants for the treatment of atherosclerosis, and designed to provide a temporary scaffold that is subsequently absorbed by blood vessels over time. Presently, there is insufficient data on the biological and mechanical responses of blood vessels accompanied by bioresorbable scaffolds (BRS) degradation. Therefore, it is necessary to investigate the inflexion point of degradation, the response of blood vessels, and the pathophysiological process of vascular, as results of such studies will be of great value for the design of next generation of BRS. In this study, abdominal aortas of SD rats were received 3-D printed poly-l-actide vascular scaffolds (PLS) for various durations up to 12 months. The response of PLS implanted aorta went through two distinct processes: (1) the neointima with desirable barrier function was obtained in 1 month, accompanied with slow degradation, inflammation, and intimal hyperplasia; (2) significant degradation occurred from 6 months, accompanied with decreasing inflammation and intimal hyperplasia, while the extracellular matrix recovered to normal vessels which indicate the positive remodeling. These in vivo results indicate that 6 months is a key turning point. This "two-stage degradation and vascular characteristics" is proposed to elucidate the long-term effects of PLS on vascular repair and demonstrated the potential of PLS in promoting endothelium function and positive remodeling, which highlights the benefits of PLS and shed some light in the future researches, such as drug combination coatings design.

Application of tetrahedral -deoxyribonucleic acid electrochemistry platform coupling aptazymes and hybridized hairpin reactions for the measurement of extracellular adenosine triphosphate in plants.

Extracellular ATP (eATP) is an important biological signal transduction molecule. Although a variety of detection methods have been extensively used in ATP sensing and analysis, accurate detection of eATP remains difficult due to its extremely low concentration and spatiotemporal distribution. Here, an eATP measurement strategy based on tetrahedral DNA (T-DNA)-modified electrode sensing platform and hybridization chain reaction (HCR) combined with G-quadruplex/Hemin (G4/Hemin) DNAzyme dual signal amplification is proposed. In this strategy, ATP aptamer and RNA-cleaving DNAzyme were combined to form a split aptazyme. In the presence of ATP, this aptazyme hydrolyzes the cleaving substrate strand with high selectivity, releasing cleaved ssDNA, which are captured by the T-DNA assembled on the electrode surface, triggering an HCR on the electrode surface to form numerous linker sequences of the HCR dsDNA product. When G-quadruplex@AuNPs (G4) spherical nucleic acid enzymes (SNAzymes) with other linkers are used as nanocatalyst tags, they are captured by HCR dsDNA through sticky linkers present on the electrode surface. An amplified electrochemical redox current signal is generated through SNAzyme-mediated catalysis of H2O2, enabling easy detection of picomole amounts of ATP. Using this strategy, eATP levels released by tobacco suspension cells were accurately measured and the distribution and concentration of eATP released on the surface of an Arabidopsis leaf was determined.

Effects of plant growth regulators on transient expression of foreign gene in Nicotiana benthamiana L. leaves.

BACKGROUND: In the last decades, replicating expression vectors based on plant geminivirus have been widely used for enhancing the efficiency of plant transient expression. By using the replicating expression vector derived from bean yellow dwarf virus and green fluorescent protein as a reporter, we investigated the effects of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine, as three common plant growth regulators, on the plant biomass and efficiency of transient expression during the process of transient expression in Nicotiana benthamiana L. leaves. RESULTS: With the increase of the concentration of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine (from 0.1 to 1.6 mg/L), the fresh weight, dry weight, and leaf area of the seedlings increased first and then returned to the levels similar to the controls (without chemical treatment). The treatment with α-naphthalene acetic acid at 0.2 and 0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.4 mg/L α-naphthalene acetic acid and was increased about by 19%, compared to the controls. Gibberellins3 at 0.1-0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.2 mg/L gibberellins3 and was increased by 25%. However, the application of 6-benzyladenine led to decrease in the level of transient expression of green fluorescent protein. CONCLUSIONS: The appropriate plant growth regulators at moderate concentration could be beneficial to the expression of foreign genes from the Agrobacterium-mediated transient expression system in plants. Thus, appropriate plant growth regulators could be considered as exogenous components that are applied for the production of recombinant protein by plant-based transient expression systems.

[Regulation of extracellular ATP onchlorophyll content and fluorescence characteristics of Angelica sinensis seedlings under drought and low temperature stress].

As an important signal molecule, extracellular ATP(eATP) can regulate many physiological and biochemical responses to plant stress. In this study, the regulation of extracellular ATP(eATP) on chlorophyll content and chlorophyll fluorescence parameters of Angelica sinensis seedlings were studied under drought and low temperature stress. The results showed that all the chlorophyll content, the actual photochemical efficiency [Y(Ⅱ)], the electron transfer rate(ETR), the photochemical quenching coefficient(qP and qL) of A. sinensis leaves were significantly decreased under drought and low temperature stress, respectively. At the same time, non-photochemical quenching(NPQ and qN) were also all significantly increased, respectively. The application of eATP alleviated the decrease of chlorophyll content, Y(Ⅱ), ETR, qP and qL of A. sinensis leaves under drought and low temperature stress, and eliminated the increase of qN and NPQ. The results indicated that eATP could effectively increase the open ratio of PSⅡ reaction centers, and improve the electron transfer rate and light energy conversion efficiency of PSⅡ of A. sinensis leaves under drought and low temperature stress. It is beneficial to enhance the chlorophyll synthesis and the adaptability of PSⅡ about A. sinensis seedlings to drought and low temperature stress.

Extracellular ATP released by copper stress could act as diffusible signal in alleviating the copper stress-induced cell death.

In the present work, by using tobacco cell suspension and wheat seedlings, we studied that eATP (extracellular ATP) released by copper (Cu) stress could act as diffusible signal in alleviating the Cu stress-induced cell death. A semipermeable membrane was fixed in the middle of a plastic box to divide the box into two equal compartments (A and B, respectively). This semipermeable membrane can prevent direct cell-to-cell (or seedling-to-seedling) contact and the diffusion of the macromolecules [such as ATPase (adenosine 5'-triphosphatase)] between these two compartments. The cell suspension directly stressed with CuCl2 was placed in compartment A and was incubated with the untreated cell suspension in compartment B. Such treatment significantly increased the levels of cell death and eATP content of the cell suspension in these two compartments. In contrast, addition of ATPase into the cell suspension directly stressed with CuCl2 decreased the eATP level in these two compartments but further increased the level of cell death in compartment B, compared to no addition of ATPase. Similar results were obtained when tobacco cell suspension was replaced by wheat seedlings. These observations indicate that when Cu stress from compartment A induced the plant cell death in compartment B, ATP transferred from compartment A could play a role in alleviating this cell death. Thus, it is suggested that eATP released by copper stress could act as diffusible signal in alleviating the Cu stress-induced cell death.

Effects of extracellular ATP on local and systemic responses of bean (Phaseolus vulgaris L) leaves to wounding.

Wounding increased the extracellular Adenosine 5'-triphosphate (eATP) level of kidney bean leaves. Treatment with wounding or exogenous ATP increased the hydrogen peroxide (H2O2) content, activities of catalase and polyphenol oxidase, and malondialdehyde content in both the treated and systemic leaves. Pre-treatment with ATP-degrading enzyme, apyrase, to the wounded leaves reduced the wound-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Application of dimethylthiourea (DMTU) and diphenylene iodonium (DPI) to the wounded and ATP-treated leaves, respectively, reduced the wound- and ATP-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Moreover, the wound- and ATP-induced systemic increases of these physiological parameters were suppressed when DMTU or DPI applied to leaf petiole of the wounded and ATP-treated leaves. These results suggest that eATP at wounded sites could mediate the wound-induced local and systemic responses by H2O2-dependent signal transduction.

[Physiological effects of cadmium stress on Astragalus membranaceus seedlings and alleviative effects of attapulgite clay on cadmium stress].

In this study we investigate the effects of cadmium stress on Astragalus membranaceus seedlings and the alleviative effects of attapulgite clay in growth substrate on cadmium stress to A. membranaceus seedlings. The results showed that the Y (Ⅱ) (effective photochemical quantum yield of PSⅡ photosynthetic), qP(photochemical quenching coefficient), ETR(the rate of non-cyclic electrontransport through PSⅡ), and chlorophyll content of the leaves were significantly decreased with the increase of cadmium concentrations, while the cadmium content, non-photochemical quenching(NPQ, qN) of the leaves and cadmium content, MDA content, plasma membrane permeability, and the damage degree of root apical membrane of the roots were significantly increased. Simultaneously, the activities of APX(ascorbate peroxidase), SOD(superoxide dismutase), POD(peroxidase), CAT(catalase), soluble protein content, and soluble sugar content of roots were increased first but then decreased with the increasing cadmium concentration. Under the condition of without Cd stress, the attapulgite clay into the growth substrate did not significantly affect above physiological indexes of leaves, but significantly increased SOD activity and soluble sugar content of roots and decreased the MDA content, damage degree of root apical membrane of roots, while other physiological indexes did not significantly change. Under cadmium stress, the presence of attapulgite clay in the growth substrate significantly alleviated the cadmium-induced decreases Y (Ⅱ), qP, ETR and chlorophyll content of leaves, and the CAT activity, soluble protein content, and soluble sugar content of roots. Under condition with cadmium stress, the presence of attapulgite clay significantly alleviated the cadmium-induced increases of leaves cadmium content, qN and NPQ, and the cadmium content, MDA content, plasma membrane permeability, damage degree of root apical membrane, SOD, POD, and APX activity of the roots. And, the alleviative effects of attapulgite clay on cadmium stress to A. membranaceus roots were more obvious with the increase of cadmium stress time. The above results showed that the addition of attapulgite clay into the growth substrate has certain alleviative effect on the cadmium stress to A. membranaceus seedlings.

The responses of photosystem II and intracellular ATP production of Arabidopsis leaves to salt stress are affected by extracellular ATP.

Hypertonic salt stress with different concentrations of NaCl increased the levels of extracellular ATP of Arabidopsis leaves. And, hypertonic salt stress decreased the levels of F v /F m (the maximal efficiency of photosystem II), Φ PSII (the photosystem II operating efficiency), qP (photochemical quenching), and intracellular ATP (iATP) production. The treatment with ß,γ-methyleneadenosine 5'-triphosphate (AMP-PCP), which can exclude extracellular ATP from its binding sites of extracellular ATP receptors, caused a further decrease in the levels of F v /F m , Φ PSII, qP, and iATP production of the salt-stressed Arabidopsis leaves, while the addition of exogenous ATP rescued the inhibitory effects of AMP-PCP on Φ PSII , qP, and iATP production under hypertonic salt stress. Under hypertonic salt stress, the values of F v /F m , Φ PSII , qP, and iATP production were lower in the dorn 1-3 mutant than in the wild-type plants. These results indicate that the responses of photosystem II and intracellular ATP production to salt stress could be affected by extracellular ATP.

Extracellular ATP: a potential regulator of plant cell death.

Adenosine 5'-triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane-associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.

Expression and signal regulation of the alternative oxidase genes under abiotic stresses.

Plants in their natural environment frequently face various abiotic stresses, such as drought, high salinity, and chilling. Plant mitochondria contain an alternative oxidase (AOX), which is encoded by a small family of nuclear genes. AOX genes have been shown to be highly responsive to abiotic stresses. Using transgenic plants with varying levels of AOX expression, it has been confirmed that AOX genes are important for abiotic stress tolerance. Although the roles of AOX under abiotic stresses have been extensively studied and there are several excellent reviews on this topic, the differential expression patterns of the AOX gene family members and the signal regulation of AOX gene(s) under abiotic stresses have not been extensively summarized. Here, we review and discuss the current progress of these two important issues.

Cell death of rice roots under salt stress may be mediated by cyanide-resistant respiration.

Treatment with solutions containing high concentrations of NaCl (200 or 300 mM) induced cell death in rice (Oryza sativa L.) roots, as well as the application of exogenous hydrogen peroxide (H2O2). Moreover, the pretreatment with dimethylthiourea (DMTU), a scavenger of H2O2, partially alleviated the root cell death induced by 200 mM NaCl. These observations suggest that the cell death of rice roots under high salt stress is linked to H2O2 accumulation in vivo. NaCl stress increased the level of cyanide-resistant respiration to some extent and enhanced the transcript levels of the alternative oxidase (AOX) genes AOX1a and AOX1b in rice roots. High-salt-stressed (200 mM NaCl) rice roots pretreated with 1 mM salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, exhibited higher levels of cell death and H2O2 production than roots subjected to either 200 mM NaCl stress or SHAM treatment alone. These results suggest that cyanide-resistant respiration could play a role in mediating root cell death under high salt stress. Furthermore, this function of cyanide-resistant respiration could relate to its ability to reduce the generation of H2O2.

Effects of hydrogen sulfide on alternative pathway respiration and induction of alternative oxidase gene expression in rice suspension cells.

The toxic effects of H2S on plants are well documented. However, the molecular mechanisms reponsible for inhibition of plants by H2S are still not completely understood. We determined the effects of NaHS in the range of 0.5-10 mM on the growth of rice suspension culture cells, as well as on the expression of the alternative oxidase (AOX) gene. AOX is the terminal oxidase of the alternative pathway (AP) and exists in plant mitochondria. The results showed that H2S treatment enhanced the AP activity. During the process of H2S treatment for 4 h, the AP activity increased dramatically and achieved the peak value at a concentration of 2 mM NaHS. Then it declined at higher concentrations of NaHS (5-10 mM) and maintained a steady level. The AOX1 gene transcript level also showed a similar change as the AP activity. Interestingly, different NaHS concentrations seemed to have different effects on the expression of AOX1a, AOX1b, and AOX1c. The induction of AOX expression by low concentrations of NaHS was inferred through a reactive oxygen species (ROS)-independent pathway. At the same time, rice cells grown in culture were very sensitive to H2S, different H2S concentrations induced an increase in the cell viability. These results indicate that the H2S-induced AOX induction might play a role in inhibiting the ROS production and have an influence on cell viability.