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In our previous study, the sequence of a matrix attachment region binding protein (MBP) cDNA was cloned from the unicellular green alga Dunaliella salina. However, the nucleotide sequence of this gene has not been reported so far. In this paper, the nucleotide sequence of MBP was cloned and characterized, and its gene copy number was determined. The MBP nucleotide sequence is 5641 bp long, and interrupted by 12 introns ranging from 132 to 562 bp. All the introns in the D. salina MBP gene have orthodox splice sites, exhibiting GT at the 5′ end and AG at the 3′ end. Southern blot analysis showed that MBP only has one copy in the D. salina genome. [ABSTRACT FROM AUTHOR]

Abstract: The expression of transgenes in mammalian cells is often at a low level mainly due to position effects from the neighboring chromatin context. To improve this, we have constructed a vector pCAM, which contains chloramphenicol acetyltransferase (CAT) reporter gene cassettes, driven by SV40 early promoter and flanked by two human β-globin MARs in cis. We transfected this vector into the Chinese hamster ovary (CHO) cell line, and found that the level of CAT gene expression with MAR was effectively increased, about 5.493-fold higher than those without MARs. Moreover, the variations of CAT expression among individuals of transformants were decreased 2.670-fold. Our result also showed that MAR could increase the proportion of positive colonies in recombinants. [Copyright &y& Elsevier]

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system. [ABSTRACT FROM AUTHOR]

During the depositional period of the Middle Jurassic Shaximiao Formation, the Western Sichuan Depression was a typical foreland basin with its foredeep in the Daba Mountain front. Controlled by tectonic uplift, large amounts of terrestrial debris were transported from the mountains. A set of fluvio-incised valley successions was deposited under semi-arid to arid climatic conditions. Autogenic and allogenic controls on the fluvial sequence developed at different times and positions have rarely been described in detail, which is noteworthy here. In this study, by integrating 3D seismic data, loggings,cores and outcrops, we investigated the sequence architecture and its sedimentary infill of the Shaximiao Formation, analyzed the different controls on fluvial successions developed in the foreland basin, and provided a sedimentary evolutional model. Our results show that: ① the Shaximiao Formation can be divided into two three-order sequences, and each one is recorded by a typical sedimentary cycle of incised valley-fluvial channel-delta, with incised valleys of 20-30m thick and 6-13km wide; ② during the phase of low frequency cycles, allogenic processes, such as tectonic movements and climatic changes, primarily controlled the progradation and regradation of the fluvial system; during the phase of high frequency cycles, autogenic processes, such as vertical and lateral accretion, mainly controlled the planform geometry of the coeval fluvial system,from the upstream to the downstream direction, the downstream decreases in the stream power appears to be responsible for the decreasing trend in the incised valley dimensions;and ③ a sedimentary model is provided to be used as a reference for the interpretation of similar fluvial systems in foreland basins. [ABSTRACT FROM AUTHOR]

Licorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored. To clarify the metabolism of liquiritigenin and liquiritin in mice. In this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching. A total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation. This study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice. Image 1 • Four LC-Q/TOF-MS/MS systems were respectively developed for mouse plasma, bile, urine and feces. • In vivo metabolites of liquiritigenin and liquiritin in mice were first studied. • A total of 26 and 24 metabolites of liquiritigenin and liquiritin in mice were identified. • Oxidation and then methylation, glucuronidation and sulfation are the main metabolism pathways. • Flavone or flavonol and its derivatives are the major metabolites of flavonones. [ABSTRACT FROM AUTHOR]

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A., (© 2024 Wiley‐VCH GmbH.)

The human cytomegalovirus major immediate early gene (CMV) promoter is currently the most preferred promoter for recombinant therapeutic proteins (RTPs) production in CHO cells. To enhance the production of RTPs, five synthetic enhancers including multiple transcription factor regulatory elements (TFREs) were evaluated to enhance recombinant protein level in transient and stably transfected CHO cells. Compared with the control, four elements can enhance the report genes expression under both two transfected states. Further, the function of these four enhancers on human serum albumin (HSA) were investigated. We found that the transient expression can increase by up to 1.5 times, and the stably expression can maximum increase by up to 2.14 times. The enhancement of transgene expression was caused by the boost of their corresponding mRNA levels. Transcriptomics analysis was performed and found that transcriptional activation and cell cycle regulation genes were involved. In conclusion, optimization of enhancers in the CMV promoter could increase the production yield of transgene in transfected CHO cells, which has significance for developing high-yield CHO cell expression system., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tian-yun Wang reports was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins., (© 2024. The Author(s).)

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells., (© 2023 Wiley-VCH GmbH.)

We have previously developed a non-viral episomal vector based on matrix attachment region (MAR) that can facilitate plasmid replication episomally in mammal cells. In this study, we have focused on the development of an alternative tissue specific episomal vector by incorporating into cis -acting elements. We found that AAT promoter demonstrated the highest eGFP expression level in HepG2, Huh-7 and HL-7702 hepatic cells. Furthermore, hCMV enhancer when combined with AAT promoter significantly improved the eGFP expression level in the transfected HepG2 cells. The mean fluorescence intensity of eGFP in hCMV2 group was 1.33 fold, which was higher than that of the control ( p  < 0.01), followed by the hCMV1 group (1.21 fold). In addition, the percentages of eGFP-expressing cells in hCMV1 and hCMV2 groups were observed to be 49.3% and 57.2%, which were significantly higher than that of the enhancer-devoid control vector (44.3%) ( p  < 0.05). Moreover, the eGFP protein were up to 3.5 fold and 5.1 fold ( p  < 0.05), respectively. This observation could be related with the activities of some specific transcription factors (TFs) during the transcriptional process, such as SRF, REL and CREB1. The composite CMV/AAT promoter can be thus used for efficient transgene expression of MAR-based episomal vector in liver cells and as a potential gene transfer tools for the management of liver diseases., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03774-x., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: In July, 2022, an increase in diphtheria cases caused by toxigenic Corynebacterium diphtheriae (C diphtheriae) was reported among asylum seekers arriving by small boats to England. Rising case numbers presented challenges for case and contact management in initial reception centres, prompting changes to national guidance and implementation of population-based control measures. This study aimed to describe the outbreak of toxigenic C diphtheriae among asylum seekers arriving by small boats to England during 2022 by use of national surveillance data., Methods: We undertook a descriptive epidemiological analysis of cases of toxigenic C diphtheriae among asylum seekers arriving by small boats to England during 2022, incorporating genomic sequencing data, antibiotic susceptibility testing results, and epidemiological data obtained through the UK Health Security Agency's national enhanced surveillance programme. Health Protection Teams conducted risk assessments, and operational data (including details regarding offer and uptake of antibiotics and vaccinations) were obtained from National Health Service partners supporting the intervention programme., Findings: In 2022, C diphtheriae isolates from 86 asylum seekers arriving by small boats were submitted to the National Reference Laboratory for confirmation and testing. Toxigenic C diphtheriae was confirmed for 72 (84%) cases and one individual with typical diphtheritic lesions but from whom no C diphtheriae was isolated from clinical swabs was also included as a probable case, resulting in 73 cases of diphtheria. 71 (97%) were male, 39 (53%) were younger than 18 years, and 36 (49%) presented with cutaneous diphtheria. The prevalence of diphtheria was highest among Afghans (1·3%) compared with all other nationalities (<0·1%). Local antibiotic susceptibility testing identified six cases with a macrolide resistant strain., Interpretation: The increase in diphtheria coincided with a high volume of asylum seekers arriving by small boats to England during 2022, and subsequently increased clinical awareness of the disease among this population. Long-term disruption to vaccination programmes in origin countries along with barriers to accessing health care along migrant routes puts asylum seekers arriving by small boats at risk of disease. With arrivals expected to continue in 2023, the UK Health Security Agency has recommended continuation of population-based control measures in England until October, 2023, subject to ongoing review., Funding: The UK Health Security Agency., Competing Interests: Declaration of interests The UK Health Security Agency diphtheria reference laboratory is part of a WHO Collaborating Centre for reference and research on diphtheria and has received contracts from WHO to provide laboratory training, advice, and reagents to overseas laboratories. We declare no other competing interests., (Crown Copyright © 2023 Published by Elsevier Ltd. This is an open access article under the Open Government License (OGL) (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/).)

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for production of recombinant therapeutic proteins (RTPs). Small-molecule additives related to cell cycle apoptosis and autophagy regulation have been used to promote RTP production. By combining two small-molecule additives, positive synergistic effects on transgene expression were observed in CHO cells. In the present study, six small-molecule additives were used, including hydrocinnamic acid (HCA), sodium butyrate (NaB), lithium acetate (LiAc), sodium succinate dibasic hexahydrate (SDH), decitabine (DAC), and sodium propionate (SP). Experiments to test the effects of their pairwise combinations on two different recombinant CHO cell lines (rCHO) were designed using Design-Expert 12.0. Different effects of various pairs of small molecules on apoptosis- and autophagy-related protein expression were observed in the rCHOs. The results showed that compared to the control culture, NaB alone increased the volumetric yield and specific productivity (Qp) by 166% and 143%, respectively. The volumetric yield and Qp of NaB combined with DAC (Cg1)-treated cells increased by 178% and 212%, respectively. Cg1 selectively blocked the cells in the G0/G1 cell cycle stage. The relative expression levels of B-cell lymphoma 2 (Bcl-2), Beclin 1, and microtubule-associated protein light chain 3 (LC3B) in Cg1-treated CHO cells were significantly increased, while relative levels of cleaved caspase-3 expression were significantly decreased. In conclusion, Cg1 had the most obvious effect on RTP production and Qp in CHO cells, suggesting the Cg1 combination of small molecules may be used to improve the expression of recombinant protein in CHO cells., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the β-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080 cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080 cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080 cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080 cells., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.

Objectives: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism., Results: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers., Conclusions: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (K m ) value of genistein was obvious increased while its maximal velocity (V max ) and intrinsic clearance (CL int ) values were significantly decreased by diabetic RLMs, and the V max and CL int values of kaempferol and isorhamnetin were notably enhanced while their K m values were markedly reduced. For the half-time (t 1/2 ) values of four target compounds and the K m , V max and CL int values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Because infection can thwart burn healing, microorganisms, their susceptibility patterns, and the effect of tangential excision timing on outcomes of burn patients were examined., Methods: A prospective, observational study was conducted that involved 318 patients with deep second-degree burns from a gas explosion treated in Xinxiang, Henan, China between January 2009 and December 2016. Patient demographic data, culture and antimicrobial susceptibility test results, and outcome variables (resuscitation fluid volume, signs of shock, body temperature, heart rate, and time to wound healing) were analyzed. Outcomes were compared among patients who had early (<24 hours), middle (2 to 7 days), and late (> 7 days) post burn excision., Results: Bacterial culture and drug sensitivity data were available for 314 of the 318 persons with burns >10% of total body surface area (TBSA). Of the 486 bacterial isolates, 330 (67.9%) were gram-negative and 156 (32.1%) were gram-positive. The number of isolates and resistance to third-generation cephalosporins increased over time. Patients having early tangential excision had significantly lower heart rate (P <.05) and reduced time to healing (P <.01) than patients in the middle or late excision group., Conclusion: Early tangential excision was found to be safe and to facilitate healing.

Most traditional post-electrophoretic processes need several hours to several days to finish the whole staining process and traditional staining solutions all contain methanol, acetic acid, or phosphoric acid, which not only produce the unpleasant smell but also cause environmental pollution. Here a fixation-free, fast protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol includes only staining and quick washing steps, can be completed in 0.5 h. It has a sensitivity of 10 ng. In addition, the dye stain does not contain any acid or methanol.

Objective: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells., Methods: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR., Results: Without gMAR expression vector,the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter,but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening,the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter,flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1,the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold,respectively., Conclusion: The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene,which may be related to the increase of gene copy number., (CopyrightCopyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective: To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector., Methods: The MAR of β-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method., Results: The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence., Conclusion: Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.

Background: Vincristine (VCR) is a chemical that is widely used in tumor therapy. While long-term use can make tumor cells resistant to VCR, the underlying mechanisms of this resistance are still unclear., Objective: This study aimed at investigating the role of microRNA (miRNA) in colon cancer drug resistance., Methods: HCT-8 colon carcinoma cells were cultured and treated with different VCR concentrations to establish an HCT-8/VCR resistant cell line. Whole-genome screens, HiSeq 2500 sequencing, and bioinformatics methods were used to detect and analyze differences in miRNA expression between the drug-resistant HCT-8/VCR cells and non-resistant HCT-8 cells. Differential expression profiles of miRNAs were constructed based on sequencing result., Results: The HCT-8/VCR resistant colon carcinoma cell line was established. With regard to the difference in drug resistance between HCT-8/VCR and HCT-8 cells, 24 miRNAs showed statistically significant differences in their expression (fold change > 4), of which 17 were up-regulated. Seven miRNAs were down-regulated., Conclusion: As abnormal expression of miRNAs was associated with VCR resistance of colon carcinoma cells, differences in miRNA expression may play a key role in VCR resistance of colon cancer cells.

Aim: To investigate human epidermal growth factor receptor 2 (HER2) amplification and protein expression in mixed gastric carcinoma., Methods: Fluorescence in situ hybridization and immunohistochemistry were used to detect HER2 amplification and protein expression in 277 cases of mixed gastric carcinoma. Protein staining intensity was rate as 1+, 2+, or 3+., Results: Of the 277 cases, 114 (41.2%) expressed HER2 protein. HER2 3+ staining was observed in 28/277 (10.1%) cases, 2+ in 37/277 (13.4%) cases, and 1+ in 49/277 (17.7%) cases. A HER2 amplification rate of 17% was detected, of which 25/28 (89.3%) were observed in the HER2 3+ staining group, 17/37 (45.9%) in 2+, and 5/49 (10.2%) in 1+. Of the 47 patients with HER2 amplification who received chemotherapy plus trastuzumab, 22 demonstrated median progression-free and overall survivals of 9.1 mo and 16.7 mo, respectively, which were significantly better than those achieved with chemotherapy alone (5.6 mo and 12.1 mo, respectively) in 19 previously treated patients (Ps < 0.05)., Conclusion: HER2 detection in mixed gastric carcinoma displays high heterogeneity. Relatively quantitative parameters are needed for assessing the level of HER2 amplification and protein expression.

Nuclear DNA of eukaryotic organism attaches to the proteinaceous nuclear matrices via specific matrix attachment regions (MARs). In order to investigate the interactions between chromosomal DNA and nuclear matrices,we isolated the MARs from unicellular alga Dunaliella salina. As the first step,a random MAR library was set up and then the binding affinity of the selected clones to nuclear matrices was tested in this study. Three DNA fragments were found to bind specifically to the nuclear matrices in vitro,of which two were strong binders and all contained known consensus motifs and a hairpin loop structure of MAR.