Attachment Region Research Articles

Two-Temperature Chemical Non-equilibrium Modeling of Argon DC Arc Plasma Torch

A 2D two-temperature chemical non-equilibrium model has been developed to investigate the plasma characteristics inside a DC arc plasma torch of argon. In this study the chemical kinetic model is carefully examined in order to properly capture the spatial variations of plasma characteristics, especially near the electrodes and arc fringe, where large gradients of plasma parameters exist. Two different sets of chemical kinetic processes are adopted in the calculation. The first chemical kinetic model [model (a)] includes the ground-state argon atoms, excited argon 4s state, atomic ions, molecular ions, and electrons, while the second model [model (b)] only considers the ground-state argon atoms, excited argon 4s state, atomic ions, and electrons. The predicted exit temperature and arc voltage by model (a) are in better agreement with experimental measurements. A delayed anode attachment with a higher arc voltage occurs in model (a) due to the low electron density in the upstream region of arc-anode attachment. Compared with the wide arc-anode attachment zone of model (b), model (a) exhibits a more constricted anode arc root with higher current density, which leads to a higher heat flux and temperature on the anode. This phenomenon may be explained by the rapid loss of electrons outside the arc-anode attachment zone due to the dissociative recombination of argon molecular ions. Moreover, model (a) produces a longer arc column with slightly higher maximum temperature and velocity on the axis, which is caused by its larger input energy.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells

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-1080cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080cell 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-1080cell 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-1080cells.

Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

Four exogenous genes, Cry3A, Cry1Ac, mtlD, and BADH, were inserted into the p1870 vector to obtain multigenic transgenic Populus nigra L. with improved insect resistance and salt tolerance. During vector construction, different promoters were used for each gene, the AtADH 5′-UTR enhancer was added between the Cry1Ac promoter and the target gene, and the matrix attachment region (MAR, GenBank: U67919.1) structure was added at both ends of the vector. It was then successfully transferred into the genome of European black poplar by Agrobacterium-mediated leaf disk transformation, and a total of 28 transgenic lines were obtained by kanamycin screening. Five events with the highest insect resistance were selected based on preliminary tests: nos. 1, 7, 9, 12, and 17. PCR, real-time PCR, and enzyme-linked immunosorbent assays (ELISA) were used to detect the expression of exogenous genes and to analyze the Bt protein toxin levels in transgenic lines from June to October. PCR results showed that all four genes were successfully introduced into the five selected lines. Fluorescence quantitative PCR showed no significant differences in the transcript abundance of the four exogenous genes between different lines. A Bt protein toxin assay showed that the Cry3A protein toxin content was significantly higher than the Cry1Ac protein toxin content by approximately three orders of magnitude. Levels of the two toxins were negatively correlated. Over the course of the growing season, Cry1Ac content raised and varied between 0.46 and 18.41 ng·g−1. Cry3A content decreased over the same time period and varied between 2642.75 and 15775.22 ng·g−1. Indoor insect feeding assay showed that the transgenic lines had high insect resistance, with mortality rates of 1–2-year-old Hyphantria cunea larvae reaching more than 80%, and those of Plagiodera versicolora larvae and nymphs reaching 100%. No. 17 and no. 12 lines had better insect resistance to Lepidoptera and Coleoptera pests. There was no clear improvement in salt tolerance of the transgenic lines, but comprehensive evaluation of 11 salt tolerance indicators showed that lines no. 17 and no. 7 had certain degrees of salt tolerance.

Repeat induces not only gene silencing, but also gene activation in mammalian cells.

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32-3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5'-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production.

Concept for the calculation of the distribution of heat input in the cathode area by GMA welding

Currently used approaches for the modeling of the heat input in gas metal arc (GMA) welding process simulation usually assume an axisymmetrical Gaussian distribution of heat flux in the cathode region. However, as in GMA welding, cathodic electron emission of non-refractory metals is involved; the attachment region consists of multiple highly mobile cathode spots, which have a highly concentrated current density, which cannot be explained solely by thermionic emission, as is present in refractory cathodes. In this work, a novel concept is presented to determine the distribution of the cathode spots, allowing to determine the distribution of heat flux and current density, to serve as boundary conditions in a magneto-hydrodynamic weld pool simulation. While the concept does not yet deliver a fully convergent solution, a model lies at its core, which takes into account the experimentally determined high current density and provides a relationship between the cathode surface temperature, the generated heat flux, and the current density. By applying a stochastic cellular automaton method, the weighted random walk of the movement of the cathode spots is simulated, according to a probability determined by the potentially released heat flux. Averaging over time gives a spatially resolved distribution of heat flux and current density.

Three-dimensional chemical non-equilibrium simulation of an argon transferred arc with cross-flow

A three-dimensional chemical non-equilibrium plasma simulation is performed to investigate the plasma characteristics of a high-intensity argon transferred arc exposed to cross-flow. The model is validated by comparison with experimental data from the literature, and reasonable agreement is obtained for the calculated electron temperature distributions. The simulation results indicate that the interactions of the cross gas flow with the plasma column have a significant influence on the distributions of the electric field close to the anode surface. At a high cross-flow rate, corresponding changes to the distributions of current density and Joule heating occur, leading to a high electron temperature zone stretching upstream of the arc attachment region. By examining the chemical reaction processes near the anode surface, it is found that the rate of electron-impact ionization of excited atoms and the three-body recombination rate are comparable at the positions in the upstream region close to the anode surface where the electron temperature is high, so the electron density does not increase in this region. Compared to the case without cross gas flow, the heavy-species temperature distribution becomes more constricted at a large cross-flow rate, and the energy loss due to the convective cooling makes the temperature gradients steeper on the side facing the cross-flow.

Morphological magnetic resonance imaging study of oral submucosal tissue and buccinator muscle dynamics in the posterior dentition: A clinical study

Statement of problemThe relationship between the buccal mucosa-tongue side wall contact points and at what ratio the submucosal tissue (ST) and buccinator muscle (BUC) change during function are unclear. PurposeThe purpose of this clinical study was to clarify the space and dynamics of the ST and BUC in complete denture wearers by using magnetic resonance imaging and to investigate how denture base shape affects space sealing and the relationship between the ST and BUC. Material and methodsEight edentulous participants wearing maxillary and mandibular complete dentures were enrolled. Wax was added to the buccal border of the dentures, and axial and coronal magnetic resonance imaging scans were made during mandibular rest (MR) to observe the relationship of the buccal mucosa and tongue above the retromolar pads. In addition, on axial images, the thicknesses of the ST and BUC were measured at 3 sites: second molar center, second molar distal (SMD), and retromolar pad center (RPC). Coronal images were made during MR, partial mouth opening, and midmouth opening (MMo). At second molar center, SMD, and RPC, the thicknesses of the ST and BUC were measured at the maxillary buccinator attachment region (point A), the mandibular buccinator attachment region (point B), and the median point between A and B (point M). ResultsDuring MR, contact sealing of the buccal mucosa and tongue on the RPC was noted in 81% of participants. After expanding the denture base with wax, contact was lost in 86% of participants. The ST and BUC thicknesses on the RPC decreased significantly with the addition of wax. During MR, the ST became significantly thicker the further posteriorly it was located. The ST was significantly thicker at point M than at point A for all sections, regardless of mouth opening. The ST and BUC thicknesses in SMD and RPC were significantly thicker at point M than at point B during MR and MMo. The differences of the ST and BUC thicknesses depending on the opening amount were observed only at the point M. In the RPC, the thickness of the ST and BUC decreased significantly as the opening amount increased (ST thickness between MR and partial mouth opening, MR, and MMo: P=.007, P=.001, BUC: P=.018, P=.001, respectively) ConclusionsThe thickness of the ST and BUC differed depending on the site. During mouth opening, these changes in thickness at each site are proportional to the differences in ST and BUC thickness.

Construction of a Novel, Dual, Self-Replicating Minicircle; an Efficient Tool in Transdifferentiation

Background & Aim Minicircles (MCs) are circular DNAs contain minimal eukaryotic expression cassette (EEC) devoid of bacterial backbone (BB). Due to low-toxicity and Immunogenicity of Minicircles, these vectors could serve as an attractive alternative in therapeutic applications such as DNA vaccines, pluripotent cells or gene-based therapies. The inclusion of scaffold/matrix attachment region (S/MAR) elements in minicircles can increase long-term expression of transgene in the dividing cells. In this study we constructed a dual reporter minicircle (DR-MC) containing an S/MAR element and two different reporter genes, mCherry and EGFP, which express in different stages after transfection into eukaryotic cells. First reporter gene is flanked by two Lox recognition sites and after adequate level of expression removes by the intramolecular Cre-madiated recombination. So, the second reporter locate adjacent to promoter and express. Reporter genes could be replaced by the gene of interests, involving in transdifferentiation. Methods, Results & Conclusion Two different fragments were amplified by PCR, loxP-mCherry-BGHpA-loxH (LML) and EGFP-SMAR-SV40pA-SV40promoter (ESS), which respectively were cloned between attB-attP sites of parental plasmid (PP-LML-ESS). Minicircle producing bacteria (ZYCY10P3S2T) were transformed with PP-LML-ESS, producing DR-MCs after induction by L-Arabinose. DR-MCs were transfected into HEK293T cells and mCherry expression was assessed by fluorescent microscopy and Flow cytometry. Tat-NLS-Cre recombinase was added to transfected cell's culture medium and EGFP expression was measured in several time points. Reporter-expressing cells were picked up to generate stable cell lines, evaluated by qRT-PCR and FISH analysis to determine the episomal state of MCs and copy numbers. The accuracy of constructed PP-LML-ESS and DR-MC was determined by restriction digestion. mCherry expression in transfected HEK293T cells confirmed the elimination of BB. Tat-NLS-Cre recombinase led to mCherry excision and EGFP expression. By tracking reporter-expressing cell lines after several passages we realized that DR-MC could replicate episomaly with maintained copy numbers. In conclusion DR-MCs propose safe, long-term and efficient systems which could carry several genes with adjustable expression time points. These characteristics enable DR-MCs to be used in cell's lineage reprogramming, in which one mature somatic cell transforms into another mature somatic cell with different gene expression profile.

Three-dimensional changes in the craniofacial complex associated with soft-diet feeding

The masticatory force affects craniofacial development. We aimed to quantify the topological deviation of the growing craniofacial structure due to soft-food diet feeding and to map the region where the phenotypes appeared on three-dimensional (3D) images. Mice were fed a powdered soft diet (SD) or conventional hard diet (HD) of regular rodent pellets at 3 weeks of age until 9 weeks of age. The heads, excluding the mandibles, were scanned by micro-computed tomography. The topographic deviation of the bony surface was quantitatively assessed by a wire mesh fitting analysis. The actual displacement and significant differences were mapped and visualized in each x-, y-, and z-axis on the 3D craniofacial image. On these reconstructed images, two-dimensional linear measurements between the landmark points confirmed the 3D skeletal displacement. In the transverse direction, the zygomatic arches and the region in which the temporal muscle attaches to the parietal and temporal bones were narrow in the SD group. The temporal muscle attachment regions significantly shifted anteriorly, and consequently, the sagittal zygomatic arch shortened. Although the cranial sagittal length was not affected, the vertical height was also reduced in the SD group compared to the HD group. Our 3D surface-based analysis demonstrated that SD feeding resulted in reduced 3D bony development at the region where the chewing muscles attach to the zygomatic arches and the temporal and parietal bones. Interestingly, SD feeding induced an anterior shift in the temporal and parietal bone regions, which can affect the skeletal inter-jaw relationship.

The M4 insulator, the TM2 matrix attachment region, and the double copy of the heavy chain gene contribute to the enhanced accumulation of the PHB-01 antibody in tobacco plants.

The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.

Comperative evaluation of the current and new design miniplate fixation techniques of the advanced sagittal split ramus osteotomy using three-dimensional finite element analysis.

The aim of this study was to evaluate the stress occurring in the fixation systems both developed in various geometric designs for this study and currently used in sagittal split ramus advancement osteotomy using finite element analysis. The finite element model that imitates three-dimensional sagittal split advancement osteotomy was fixed in 10 different miniplate fixation methods: one miniplate fixed with four monocortical screws in a horizontal and oblique pattern; four-hole two miniplates with eight monocortical screws; five-hole miniplate fixed with four monocortical and one bicortical screws; six-hole straight and curved miniplates fixed with six monocortical screws in different geometric designs. Unilateral masticatory muscle loads that have previously determined in the literature were applied to the model at the anatomical muscle attachment regions and the data obtained from finite element analysis and static linear analysis methods were recorded as Von mises, maximum principle and minimum principle stress values. It was observed from the results that maximum stress occurred in Group 1, which consisted of double backward T-shaped miniplate with 6-holes and, minimum stress occured in group 10, which mimiced hybrid system with one miniplate and four monocortical and one bicortical screws. Based on our results, the stress on the miniplates changes according to the geometric designs and the stress on the miniplate decreases as the numbers of miniplates and bars increase. The hybrid miniplate may be preferred by the surgeon as it will be exposed to less stress in excessive mandibular advancements by using the advantages of both the miniplate and the bicortical screw.

FoxO1 is a cell-specific core transcription factor for endometrial remodeling and homeostasis during menstrual cycle and early pregnancy.

Endometrium is a vital multicellular tissue for progression of pregnancy. Forkhead box O1 (FoxO1) transcription factor plays an important role in the endometrium as it regulates various cellular processes with its unique expression in different cell types. This review focuses on the role of FoxO1 in endometrium with a particular emphasis on FoxO1 signaling in individual endometrial cell types during the menstrual cycle and early pregnancy. A literature search was conducted in PubMed, Web of Science and Scopus to select studies reporting the role of FoxO1 in endometrium using the keywords: FoxO1, endometrium, menstrual cycle, early pregnancy, endometrial receptivity, implantation, decidualization, angiogenesis and neoplasia. Papers published before October 2020 were selected. Drawing on advances in laboratory science and preclinical studies, we performed a narrative review of the scientific literature to provide a timely update on the roles of FoxO1 during the menstrual cycle and early pregnancy. FoxO1 is considered to be a decidualization marker in endometrial stromal cells, mainly because it regulates the transcription of decidual prolactin and insulin-like growth factor-binding protein 1 genes. Importantly, 507 of 3405 genes that are specifically regulated during decidualization of human endometrial stromal cells are expressed abnormally as a result of FOXO1 reduction. Epithelial FoxO1 is currently accepted as a novel endometrial receptivity marker for humans and mice owing to its timely and specific expression at the window of implantation. On the other hand, FOXO1 is essential in endometrial epithelial cell proliferation and differentiation to achieve endometrial homeostasis since loss of function of FOXO1 causes endometrial neoplasia. Last but not least, FoxO1 seems to act like a navigator molecule for embryo homing owing to its notably decreased nuclear expression in endometrial luminal epithelial cells, specifically at the blastocyst attachment region, which results in differentiation, entosis and apoptosis of endometrial epithelial cells during the peri-implantation period. In endothelium, FoxO1 expression coincides with the timing of increased vascular permeabilization during early pregnancy. There are limited data regarding the importance of FoxO1 upregulation in endometrial endothelial cells, therefore, it is time to investigate the role of endothelial FoxO1, which is the missing piece in the puzzle of the enigmatic endometrium. Another missing piece in the puzzle for the role of FoxO1 is on embryo development. FoxO1 is a cell-specific core transcription factor involved in efficient endometrial remodeling during the menstrual cycle and early pregnancy. A better understanding of the role of FoxO1 as a decidualization marker, as an emerging marker of endometrial receptivity, and as a therapeutic target to prevent endometrial neoplasia could help us to make sense of endometrial biology and thus to improve the outcomes of ART in the clinic.

Episomal vectors based on S/MAR and the \u03b2-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient \u03b3-globin activation in haematopoietic cells

We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

Interactions of adjuvants on adhesion and germination of Isaria fumosorosea on adults of Diaphorina citri

ABSTRACT: Asian citrus psyllid, Diaphorina citri, is considered the most important citrus pest worldwide, as it transmits Huanglongbing – serious citrus disease. New efficient and sustainable strategies to control this pest have been investigated and the use of entomopathogenic fungi has become a promising alternative. The objective of this study was to evaluate the effects of adjuvants a) Tween 80 at 0.01 % (v/v); b) Silwet L77 at 0.025 % (v/v) and c) KBRAdj at 0.075 % (v/v) on adhesion, germination and pathogenicity of Isaria fumosorosea ESALQ-1296(5 × 106 conidia mL–1). Female adults of D. citri used in this experiment were sprayed on Citrus limonia seedlings. The sprayed insects were analyzed through scanning electron microscopy (SEM) to identify the most susceptible integument regions for fungus attachment and the effect of adjuvants used. In the pathogenicity test, adjuvants Silwet L77 and KBRAdj presented a higher efficiency than Tween 80. Fungi adhered predominantly to the ventral posterior (abdomen) region in comparison with the dorsal anterior (thorax) region. In addition, adjuvants Silwet L77 and KBRAdj presented faster germination ( 72 h). Conidial germination in the dorsal part of the thorax of the insects was observed only with adjuvant KBRAdj 72 h post inoculation.

Distance effect characteristic of the matrix attachment region increases recombinant protein expression in Chinese hamster ovary cells.

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. Compared with the control vector, 500 and 1000bp 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 2500bp spacer DNAs were inserted, but a declining trend was observed when a 1500bp spacer DNA was inserted. The vector containing a 500bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. 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.

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.

Enhanced transgene expression using two β-globin MARs flanking expression cassettes in stably transfected CHO-K1 cells.

In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human β-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5' site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.

Numerical simulation of constricted and diffusive arc–anode attachments in wall-stabilized transferred argon arcs

A numerical simulation is conducted to investigate arc–anode attachment behavior, especially the formation mechanism of the constricted arc attachment mode for the water-cooled anode of wall-stabilized transferred argon arcs. Argon molecular ions and the corresponding kinetic processes are included to the finite-rate chemistry model in order to capture the chemical nonequilibrium characteristics of the arc near the anode region. Modeling results show that constricted and diffusive arc–anode attachments can be self-consistently obtained at different arc currents while keeping other parameters unchanged. The dominant kinetic processes contributing to ionization and recombination in the arc center and fringes are presented. The results show that in arc fringes and the arc attachment region, molecular ion recombination plays an important role which leads to the rapid loss of electrons. The radial evolution of the production, loss and transport processes of electrons is further analyzed. It is found that for the constricted arc attachment mode, both the recombination and convection transport caused by the anode jet result in the loss of electrons at the arc fringes, which leads to the shrinkage of the arc column at the anode. The formation of the anode jet is due to the combined action of radial and axial Lorentz forces in the anode region.

Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells.

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.

Effect of inflow swirl on the vane endwall aerothermal performance with endwall misalignment at transonic condition

Effect of turbine inlet swirl condition (swirl intensity and orientation) on endwall heat transfer performance with combustor-turbine misalignment at transonic condition was numerically investigated, and coolant injection from combustor-turbine interface was also considered. Simulations with two mainstream swirl numbers (SN) of 0.6 and 1.0, two swirl orientations, two endwall misalignment modes and slot coolant injection were conducted by solving the three-dimensional RANS equations coupled with the Reynolds Stress Turbulence Model (RSM). Computational results indicate that for anticlockwise swirled condition, the averaged Nu number of the high heat transfer attachment region upstream of the leading edge decreases from 4400 to 3100 with the increasing SN from 0.6 to 1.0. Negative incidence is caused at hub followed by a low heat transfer region at pressure side junction. Enhanced horseshoe vortex and mainstream direct attachment enlarge the hot ring (uncooled area around the vane leading edge) when SN = 0.6. As for clockwise swirled condition, positive incidence is caused at hub and can have some effect on reducing the passage crossflow. The pressure side low heat transfer region enlarges under aligned mode but shrinks under misaligned mode compared with anticlockwise condition. Averaged Nu distributes more uniformly along axial direction. The hot ring can only be observed when SN = 0.6 under misaligned condition. For aligned mode, endwall heat transfer at x/Cax < 1.0 is 30–50% higher for SN = 0.6 than SN = 1.0. When coolant is introduced at the combustor-turbine interface, the heat transfer strength at attachment region is largely reduced. Under aligned condition, coolant tends to eject out between two swirl cores. The majority of the coolant is constrained in the recirculation region by cavity vortices under misaligned mode. Averaged Nu number at the attachment region is 4450 and 3600 under aligned and misaligned mode, respectively. But the averaged Nu number downstream of the passage throat is less than 2000 and distributes uniformly.