Characteristic analysis of fibracel carriers for animal cell culture / 中国生物制品学杂志

@#Objective To investigate the surface properties of different fibracel carriers and their culture effects on different cells.Methods Three fibracel carriers(A,B,C)were selected to analyze the chemical element composition of their materials,and the contact angles of the carriers before and after pretreatment with 0. 1 mol/L NaOH solution were tested. By measuring the adhesion effect and glucose consumption of Vero,MDCK and MRC-5 cells on the carrier,and observing the cell growth state by fluorescent staining,the cell adhesion efficiency and culture effect of the three carriers were compared and analyzed.Results The three carriers were mainly composed of C,H,O,and contained a small amount of N and S elements. Before pretreatment,the contact angle of carrier B was 0°,which was significantly lower than that of A[(109 ± 3. 13)°]and C[(121 ± 6. 82)°]（each F = 709. 1,each P < 0. 000 1）,and the hydrophilicity was stronger. Carriers A and C had poor hydrophilicity. After pretreatment,the contact angles of the surfaces of the three carriers A,B,and C were all 0°,with no significant difference(F = 0. 069 4,P > 0. 05),all of which were hydrophilic. The adherence rates of the three types of carriers within 3 h of cell culture were all above 80%. The cells were dense and evenly grown on the carrier fibers,the glucose consumption curves tended to“S”type,and the continuous cell culture effect was good. The total glucose consumption of carrier A and carrier C was basically the same,and carrier B was lower than carrier A and carrier C.Conclusion The chemical element composition and the relationship between the hydrophilic and hydrophobic properties of the three fibracel carriers were analyzed,and the adhesion rate and culture effect of Vero,MDCK and MRC-5 cells were evaluated,which provide reference for the subsequent research and production application of fibracel carriers.

Exploring the micromorphological characteristics of adult lower cervical vertebrae based on micro-computed tomography.

We will use micro-computed tomography to scan 31 sets of the adult lower cervical vertebrae (155 vertebrae) to observe the morphological characteristics and direction of trabeculae in the lower cervical vertebrae by outlining and reconstructing the regions of interest and to calculate the variation laws of the microstructure in the regions of interest to reveal their structural characteristics and weak areas. As a result, the images showed that the trabeculae in the lower cervical pedicle near the medial and lateral cortices were relatively dense, and their bone plates were lamellar. There were cavities between the superior and inferior articular processes where the ossification centers had not been absorbed after ossified. The lamellar trabeculae in the vertebral plates near the cortical bones were only 1-2 layers, extended and transformed into rod-shaped trabeculae in a radial shape toward the medullary space. The lamellar trabeculae of the vertebral plate extend over the spinous process near the cortical bone. The statistical results of the trabeculae's morphological parameters showed significant differences in bone volume fraction values among the four parts (P < 0.05). There were substantial differences in BS/BV, except for no differences between the pedicle and the vertebral plate (P < 0.05). There was a significant difference in trabecular pattern factor values between the articular process, the spinous process, and the vertebral plate (P < 0.05) and a significant difference between the pedicle, the spinous process, and the vertebral plate (P < 0.05). There were no significant differences in trabecular bone thickness and trabecular space values among the four parts (P < 0.05). The anatomical microstructural perspective confirms that the optimal choice is internal fixation via the pedicle. If using pedicle screws, the nail tract needs to be placed into the spinous process to increase its holding power and resistance to extraction.

Can the digital economy improve green total factor productivity? An empirical study based on Chinese urban data.

With the new generation of technological revolution, the digital economy has progressively become a key driver of global economic development. In this context, how to promote green economic growth and improve green total factor productivity (GTFP) with the help of the digital economy is an important issue that urgently needs empirical research. We adopted the panel data of 278 Chinese prefecture-level cities from 2011 to 2020 to test whether the digital economy improves the GTFP through the Gaussian Mixed Model (GMM) dynamic panel model. The moderating effect model has been used to explore the impact mechanism from the perspectives of industrial structure upgrade and environmental regulation. In addition, a grouping regression was applied to the sample cities to test the heterogeneous impact of the digital economy on the GTFP. Based upon the empirical findings, this work has the following conclusions. First, the digital economy plays a significant role in improving the GTFP. Second, an industrial structure upgrade has a positive moderating effect on the ability of the digital economy to enhance the GTFP. The environmental regulation, in contrast, has a negative moderating effect. Third, the digital economy exerts heterogeneous impacts on the GTFP across regions, but not at the city level.

Advances in research and application of animal cell culture matrix fibracel carriers / 中国生物制品学杂志

@#Fibracel carriers based on polyester fiber have the advantages of good acid and alkali resistance,heat resistance,good biocompatibility,non-biodegradation,and can promote cell adhesion and growth. With no animal-derived ingredients and high biological safety,it is one of the preferred carriers for cell culture matrixes,which has been widely used in the development and production of cell matrix biological products with the development of biotechnology in recent years. This paper reviewed the structure and cell culture characteristics of fibracel carriers as well as the applications in vaccine production,cell therapy and tissue engineering,so as to provide a theoretical basis for the further development and application of fibracel carrier technology.

Examination of the microstructures of the lower cervical facet based on micro-computed tomography: A cadaver study.

The cervical facet has complicated 3D microstructures and inhomogeneities. The cervical facet joint, which also participates in the formation, plays a certain role in regulating and limiting the movement of the spine. Correct identification and evaluation of its microstructure can help in the diagnosis of orthopedic disease and predict early phases of fracture risk. To evaluate the safety of the cervical spine by measuring and analyzing the microstructures and morphometric parameters of bone trabeculae in the normal cervical facet with high-resolution 3D micro-computed tomography. Thirty-one sets of C3 to C7 lower cervical vertebrae (155 vertebrae) were scanned using micro-computed tomography. The morphological characteristics and direction of trabecular bone in the facet of the lower cervical vertebrae were observed by selecting and rebuilding the areas of interest, and the changes in the microstructure of the areas of interest were calculated to reveal the structural characteristics and weak areas. Images indicated an ossified center between the superior and inferior articular processes of the lower cervical spine. The cellular bone trabeculae of the articular process had complex reticular microstructures. The trabecular bone plate near the cortical bone was lamellar and relatively dense, and it extended around and transformed into a network structure, and then into the rod-shaped trabecular bone. The rod-shaped trabeculae converged with the plate-shaped trabeculae with only 1 to 2 layers surrounding the trabeculae cavity. Statistical results of the morphological parameters of the trabecular bone showed that trabecular bone volume fraction values were significantly higher for C7 than for C3 to C6 (P < .05). There were significant differences between C7 and C3 to C5 and between C6 and C4 in bone surface area/bone volume (P < .05). There was a significant difference between C7 and C3 to C6 in trabecular bone thickness values (P < .05). The degree of anisotropy value was significantly smaller for C3 than for C6 and C7 (P < .05). The changes in the C3 to C7 microstructure were summarized in this study. The loading capacity and stress of the C7 articular process tended to be limited, and the risk of injury tended to be higher for the C7 articular process.

Brassinolide Soaking Reduced Nitrite Content and Extended Color Change and Storage Time of Toona sinensis Bud during Low Temperature and Near Freezing-Point Temperature Storage.

Low temperatures are often used to preserve fruits and vegetables. However, low-temperature storage also causes problems, such as chilling injury, nitrite accumulation, and browning aggravation in plants. This study investigated the effects of brassinolide (BR,1.0 mg L-1) solution soaking, storage temperatures (-2 ± 0.5 °C, 4 ± 0.5 °C, and 20 ± 1 °C), and their combinations on nitrite content, color change, and quality of stored Toona sinensis bud. The results showed that low temperature (LT, 4 ± 0.5 °C) and near freezing-point temperature (NFPT, -2 ± 0.5 °C) storage effectively inhibited the decay of T. sinensis bud compared to room temperature (20 ± 1 °C, the control). The combined treatments of BR with LT or NFPT reduced nitrite content and maintained the color and the contents of vitamin C, carotenoids, saponins, ß-sitosterol, polyphenol, anthocyanin, flavonoids, and alkaloids in T. sinensis bud. BR soaking delayed the occurrence of chilling injury during NFPT storage. Meanwhile, BR soaking enhanced the DPPH radical scavenging activity, ABTS activity, and FRAP content by increasing SOD and POD activity and the contents of proline, soluble, and glutathione, thus decreasing MDA and hydrogen peroxide content and the rate of superoxide radical production in T. sinensis bud during NFPT storage. This study provides a valuable strategy for postharvest T. sinensis bud in LT and NFPT storage. BR soaking extended the shelf life during LT storage and maintained a better appearance and nutritional quality during NFPT storage.

Production of Atosiban's Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process.

The key intermediate NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH of Atosiban was prepared from N-Boc-S-Bzl-cysteine by the stepwise lengthening of the chain according to the repetitive N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) strategy. This synthetic route required no chromatography purification and can be readily performed, yielding a highly pure pentapeptide compound.

Melatonin alleviates aluminum-induced growth inhibition by modulating carbon and nitrogen metabolism, and reestablishing redox homeostasis in Zea mays L.

Melatonin, a regulatory molecule, performs pleiotropic functions in plants, including aluminum (Al) stress mitigation. Here, we conducted transcriptomic and physiological analyses to identify metabolic processes associated with the alleviated Al-induced growth inhibition of the melatonin-treated (MT) maize (Zea mays L.) seedlings. Melatonin decreased Al concentration in maize roots and leaves under Al stress. Al stress reduced the total dry weight (DW) by 41.2% after 7 days of treatment. By contrast, the total DW was decreased by only 19.4% in MT plants. According to RNA-Seq, enzyme activity, and metabolite content data, MT plants exhibited a higher level of relatively stable carbon and nitrogen metabolism than non-treated (NT) plants. Under Al stress, MT plants showed higher photosynthetic rate and sucrose content by 29.9% and 20.5% than NT plants, respectively. Similarly, the nitrate reductase activity and protein content of MT plants were 34.0% and 15.0% higher than those of NT plants, respectively. Furthermore, exogenous supply of melatonin mitigated Al-induced oxidative stress. Overall, our results suggest that melatonin alleviates aluminum-induced growth inhibition through modulating carbon and nitrogen metabolism, and reestablishing redox homeostasis in maize. Graphical Abstarct.

Melatonin enhances drought stress tolerance in maize through coordinated regulation of carbon and nitrogen assimilation.

Melatonin is a pleiotropic regulatory molecule in plants and is involved in regulating plant tolerance to drought stress. Here, we conducted transcriptomic and physiological analyses to identify metabolic processes associated with the enhanced tolerance of the melatonin-treated maize (Zea mays L.) seedlings to water deficit. Maize seedlings were foliar sprayed with either 50 µM melatonin or water and exposed to drought stress for 12 d in growth chambers. Drought stress significantly suppressed seedling growth, and melatonin application partially alleviated this growth inhibition. RNA-Seq analysis revealed that genes whose expression was significantly altered by melatonin were mainly related to carbon (C) and nitrogen (N) metabolism. Analysis of transcriptomics, enzyme activity, and metabolite content data, melatonin-treated plants exhibited a higher level of relatively stable C and N metabolism than untreated plants; this phenotype of melatonin-treated plants was associated with their higher photosynthesis, sucrose biosynthesis, N assimilation, and protein biosynthesis capacities under drought stress. Overall, our results suggest that melatonin enhances drought stress tolerance in maize through coordinated regulation of C and N metabolism.

A novel method for preparing Eligulstat through chiral resolution.

Eliglustat is a ceramide glucosyltransferase inhibitor work as first line oral therapy for adults with Gaucher disease type 1 (a rare disease) at present. Although the eliglustat in enantiomerically pure forms is obtained by asymmetric syntheses, the reported methods suffer from many limits when it comes to industrial applications. Therefore, the preparation of a racemic mixture followed by resolution can still be a viable and straightforward alternative, especially when it could be adapted to large scale. Herein, we developed an effective and practical synthetic route to prepare stereoisomers mixture of eliglustat, and a novel chiral resolution method to prepare eliglustat. Using 1,1'-Binaphthyl-2,2'-diyl -hydrogenphosphate (BNDHP) as resolution reagent, optical pure eliglustat (e.e. >99%, 13.97% total yield) could be obtained after recrystallization.

A Practical and Total Synthesis of Pasireotide: Synthesis of Cyclic Hexapeptide via a Three-Component Condensation.

Pasireotide is a multi-receptor ligand somatostatin analogue approved for medical treatment of Cushing's disease and acromegaly. The liquid-phase total synthesis of pasireotide-a 18-membered cyclic hexapeptide-was achieved by the 3 + 2 + 1 strategy, and the Pro1-Phe6 peptide bond was selected as the final cyclization position. Two key fragments were simply synthesized using N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) as coupling agents, and processes of the two key fragments were simple without any chromatographic purification. The current synthesis method is easily scalable and produces the target peptide with an overall yield of 15%.

Ag/Pyridine Co-Mediated Oxidative Arylthiocyanation of Activated Alkenes.

An efficient Ag/pyridine co-mediated oxidative arylthiocyanation of activated alkenes via radical addition/cyclization cascade process was developed. This reaction could be carried out under mild conditions to provide biologically interesting 3-alkylthiocyanato-2-oxindoles in good to excellent yields. Mechanistic studies suggested a unique NCS• radical addition path and clarified the dual roles of catalytic pyridine as base and crucial ligand to accelerate the oxidation of Ag(I) to Ag(II), which is likely oxidant responsible for the formation of NCS• radical. These mechanistic results may impact the design and refinement of other radical based reactions proceeding through catalytic oxidations mediated by Ag(I)-pyridine/persulfate. The chemical versatility of thiocyanate moiety was also highlighted via SCN-tailoring chemistry in post-synthetic transformation for new S-C(sp³/sp²/sp), S-P, and S-S bonds constructions. The protocol provides an easy access to many important bioisosteres in medicinal chemistry and an array of sulfur-containing 2-oxindoles that are difficult to prepare by other approaches.

A Facile Method to Prepare a Hydrophilic/Hydrophobic Metal Surface by Peptide.

A facile method to prepare a hydrophilic/hydrophobic metal surface by metal-binding peptide was proposed in this article. Metal-binding peptide sequenced NLNPNTASAMHV was taken as the target peptide to interact with stainless steel. The surface morphology, roughness and Fourier-Transform Infrared spectroscopy (FTIR) showed that some changes occurred on the modified stainless steel surface. Not only were the surfaces coarser but also some organic groups appeared on the modified sample surfaces. By comparing the CAs of all the samples, the most suitable concentration of peptide and treating time were determined. A new and facile way to endow some metals surfaces with hydrophobicity or hydrophilicity has been developed, which is useful especially for antibiofouling.

Water-Soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as an Effective Gene Delivery Vector.

Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. In this study, a series of polyphosphazenes (PPZ) bearing cyclic polyamine and imidazole groups were synthesized and investigated for gene delivery. Agarose gel electrophoresis assays showed that poly(imidazole/1,4,7,10-tetraazyclodocane)phosphazene (Im-PPZ-cyclen) had good binding ability with plasmid DNA (pDNA), yielding positively charged particles with a size around 120-140 nm from a ratio of 10:1 to 5:1 (Im-PPZ-cyclen/pDNA, w/w). The cytotoxicity of Im-PPZ-cyclen assayed by MTT was lower than that of PEI 25 kDa, and was similar to that reported for poly(di-2-dimethylaminoethylamine)phosphazene (poly(di-DMAEA)phosphazene) to some degree. The maximum transfection efficiency of Im-PPZ-cyclen/pDNA complexes against 293 T cells at the ratio of 5:1 (Im-PPZ-cyclen/pDNA, w/w) is close to that of Lipofectamine 2000. The present work may provide a strategy for the design of new cationic polymers with reduced cytotoxicity and be applied to gene delivery as an efficient nonviral vector.

Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

Insight into DNA Minor Groove Unspecific Binding of Pyrrole Polyamide.

Chemistry-based approaches have exploited base-pairing for sequence-specific recognition of DNA. A variety of sequence-specific Py-Im hairpin polyamides to target sequences of biological interest have been widely developed. Here we reported that an eight-ring N-methylpyrrole polyamide can induce a strong negative signal when it interacted with ct-DNA in the minor groove, which differs from the typical CD signal induced by hairpin polyamide reported previously. Our current efforts mainly focused on investigating possible reasons and binding mode by CD spectroscopy, singular value decomposition, and atomic force microscopy. The results suggested that partly compacted DNA may form due to the unfolded binding mode that made DNA shrink along the axis of duplex. In addition, this unfolded binding was remarkably restrained in high ionic strength medium where the neutralized phosphate groups in the DNA backbone narrowed the minor groove. The present work might help to understand deeply how the Py-Im polyamides bind to duplex DNA under different conditions and, in particular, be applied to gene manipulation and expression.

An IDB-containing low molecular weight short peptide as an efficient DNA cleavage reagent.

Artificial nucleases have attracted significant interest due to their abilities in accelerating DNA cleavage, which results in the possibility of genome manipulation. However, compared with natural nucleases, the currently available artificial nucleases have low cleavage efficiency, especially metal-free artificial nucleases. Thus, it is still a challenge to develop highly efficient metal-free artificial nucleases via a non-oxidative pathway. We here designed and prepared a group of rigid bis-amine-grafted PASP conjugates (PASP-IDB), and investigated their abilities to induce DNA double-strand cleavage. The detailed assays showed that in the absence of metal ions, these short peptide conjugates can effectively break the phosphodiester linkage at a relatively low concentration and under physiological conditions through a hydrolytic process, giving the 10(7)-fold rate acceleration over uncatalyzed double-strand DNA. The probable mechanism verified by control experiments revealed that IDBs and free carboxyl groups in PASP synergically catalyzed DNA cleavage. In addition, the effects of degrees of substitution on the cleavage activity were studied, and the results indicated the existence of minimum building blocks of PASP-IDB for efficient DNA cleavage. The results of our study have implications on the design of short peptide-based molecules as new artificial nucleases and may provide a strategy for developing safe and efficient metal-free DNA cleavage reagents.

Electrospray ionization mass spectrometric studies on the characteristic fragmentation of Asp/cyclen conjugates.

RATIONALE: Differentiation and structural characterization of Asp/cyclen conjugates by electrospray ionization tandem mass spectrometry (ESI-MS(n)) are significantly important for their biomedical application. Hence, the present study is conducted. METHODS: The fragmentations of Asp/cyclen conjugates generated by positive ion mode electrospray ionization were examined here by low-energy collision-induced dissociation (CID). ESI-MS(n) spectra of cyclen were acquired to confirm cyclen contraction products derived from the studied compounds. The fragments derived from the Asp/cyclen conjugates were proved by deuterium-exchange experiments. RESULTS: Asp/cyclen conjugates displayed characteristic dissociation pathways, including cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. It was observed that cleavages of C-terminal amide bonds generated b2 and b2 + H2O ions from the protonated CyclenAspAspAsp and a b1 + H2O ion from the protonated CyclenAspAsp. In addition, various cyclen contraction products were also observed. CONCLUSIONS: In ESI-MS(n) spectra of studied compounds, fragments of bn-1 + H2O or cyclic anhydride were generated due to facile mobilization of C-terminal or side-chain COOH protons. In addition, the cyclen contraction products were detected. These results might provide sufficient information for the identification of Asp/cyclen conjugates by mass spectrometry.

Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought.

Establishing crop cultivars with strong tolerance to P and N deprivation, high salinity, and drought is an effective way to improve crop yield and promote sustainable agriculture worldwide. A vacuolar H+-pyrophosphatase (V-H+-PPase) gene in wheat (TaVP) was functionally characterized in this study. TaVP cDNA is 2586-bp long and encodes a 775-amino-acid polypeptide that contains 10 conserved membrane-spanning domains. Transcription of TaVP was upregulated by inorganic phosphate (Pi) and N deprivation, high salinity, and drought. Transgene analysis revealed that TaVP overexpression improved plant growth under normal conditions and specifically under Pi and N deprivation stresses, high salinity, and drought. The improvement of growth of the transgenic plants was found to be closely related to elevated V-H+-PPase activities in their tonoplasts and enlarged root systems, which possibly resulted from elevated expression of auxin transport-associated genes. TaVP-overexpressing plants showed high dry mass, photosynthetic efficiencies, antioxidant enzyme activities, and P, N, and soluble carbohydrate concentrations under various growth conditions, particularly under the stress conditions. The transcription of phosphate and nitrate transporter genes was not altered in TaVP-overexpressing plants compared with the wild type, suggesting that high P and N concentrations regulated by TaVP were caused by increased root absorption area instead of alteration of Pi and NO3- acquisition kinetics. TaVP is important in the tolerance of multiple stresses and can serve as a useful genetic resource to improve plant P- and N-use efficiencies and to increase tolerance to high salinity and drought.