Reduction of metabolic waste products, ammonia and lactate, through the coupling of GS selection and LDH-A down-regulation in CHO cells

The cultivation of Chinese hamster ovary (CHO) cells for the production of therapeutic proteins inevitably accompanies the production of metabolic wastes, mostly ammonia and lactate. Ammonia alters cell growth, productivity and the glycosylation patterns of proteins, and lactate acidifies culture media, having negative effects on cell culture. A stable CHO cell line should be established for the manufacturing process of therapeutic proteins, and the development of stable cell lines is usually based on two expression systems: the dihydrofolate reductase (DHFR) system and the glutamine synthetase (GS) system. Compared to the DHFR system, the GS system produces a reduced level of ammonia because the GS protein uses ammonia to produce glutamine. In order to overcome the lactate accumulation problem, down-regulation of the lactate-producing enzyme, lactate dehydrogenase-A (LDH-A), has been shown to be effective. Engineering of the LDH-A gene has been applied for several CHO cell lines with the DHFR system, but there has been no trial which couples the ammonia reduction from the GS system and lactate reduction through cell engineering. In the present study, the GS system was used for the expression of therapeutic antibody in CHO cells, thereby reducing ammonia in the culture media. In addition, the LDH-A gene was down-regulated with shRNA to reduce lactate production. The antibody-producing cell line produces a reduced level of ammonia compared to the host cell line due to the over-expression of the GS protein. The down–regulation of the LDH-A gene in the antibody-producing cell line not only reduces the level of lactate but also further reduces the level of ammonia, accomplishing complete waste reduction. LDH-A down-regulation was also applied to the host cell lines of the GS system – the CHO-K1 cell line and the GS deficient CHO-K1 cell line. However, LDH-A down-regulated host cells could not survive the pool-selection process. Given that the GS system uses a glutamine-depleted condition as a form of selection pressure, enhanced glycolysis is inevitable and the down-regulation of LDH-A appears to hinder metabolic changes. Taken together, the application of LDH-A down-regulation in the producing cell line of the GS system successfully reduced both ammonia and lactate levels. However, LDH-A engineering could not be applied to the host cell lines because it inhibits the selection process of the GS system

Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo

Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that bepridil (BPD) and trifluoperazine (TFP), which are FDA-approved drugs for treatment of schizophrenia and angina respectively, inhibit Akt-pS473 phosphorylation and promote FOXO3 nuclear localization and activation in TNBC cells. BPD and TFP inhibit survival and proliferation in TNBC cells and suppress the growth of TNBC tumors, whereas silencing FOXO3 reduces the BPD- and TFP-mediated suppression of survival in TNBC cells. While BPD and TFP decrease the expression of oncogenic c-Myc, KLF5, and dopamine receptor DRD2 in TNBC cells, silencing FOXO3 diminishes BPD- and TFP-mediated repression of the expression of these proteins in TNBC cells. Since c-Myc, KLF5, and DRD2 have been suggested to increase cancer stem cell-like populations in various tumors, reducing these proteins in response to BPD and TFP suggests a novel FOXO3-dependent mechanism underlying BPD- and TFP-induced apoptosis in TNBC cells

Automatic 3D Registration of Dental CBCT and Face Scan Data using 2D Projection images

This paper presents a fully automatic registration method of dental cone-beam computed tomography (CBCT) and face scan data. It can be used for a digital platform of 3D jaw-teeth-face models in a variety of applications, including 3D digital treatment planning and orthognathic surgery. Difficulties in accurately merging facial scans and CBCT images are due to the different image acquisition methods and limited area of correspondence between the two facial surfaces. In addition, it is difficult to use machine learning techniques because they use face-related 3D medical data with radiation exposure, which are difficult to obtain for training. The proposed method addresses these problems by reusing an existing machine-learning-based 2D landmark detection algorithm in an open-source library and developing a novel mathematical algorithm that identifies paired 3D landmarks from knowledge of the corresponding 2D landmarks. A main contribution of this study is that the proposed method does not require annotated training data of facial landmarks because it uses a pre-trained facial landmark detection algorithm that is known to be robust and generalized to various 2D face image models. Note that this reduces a 3D landmark detection problem to a 2D problem of identifying the corresponding landmarks on two 2D projection images generated from two different projection angles. Here, the 3D landmarks for registration were selected from the sub-surfaces with the least geometric change under the CBCT and face scan environments. For the final fine-tuning of the registration, the Iterative Closest Point method was applied, which utilizes geometrical information around the 3D landmarks. The experimental results show that the proposed method achieved an averaged surface distance error of 0.74 mm for three pairs of CBCT and face scan datasets.Comment: 8 pages, 6 figures, 2 table

Premixed Calcium Silicate-Based Root Canal Sealer Reinforced with Bioactive Glass Nanoparticles to Improve Biological Properties

Recently, bioactive glass nanoparticles (BGns) have been acknowledged for their ability to promote interactions with the periapical tissue and enhance tissue regeneration by releasing therapeutic ions. However, there have been no studies on calcium silicate sealers with bioactive glass nanoparticle (BGn) additives. In the present study, a premixed calcium silicate root canal sealer reinforced with BGn (pre-mixed-RCS@BGn) was developed and its physicochemical features and biological effects were analyzed. Three specimens were in the trial: 0%, 0.5%, and 1% bioactive glass nanoparticles (BGns) were gradually added to the premixed type of calcium silicate-based sealer (pre-mixed-RCS). To elucidate the surface properties, scanning electron microscopy, X-ray diffraction, and energy-dispersive spectroscopy were used and flowability, setting time, solubility, and radiopacity were analyzed to evaluate the physical properties. Chemical properties were investigated by water contact angle, pH change, and ion release measurements. The antibacterial effects of the bioactive set sealers were tested with Enterococcus faecalis and the viability of human bone marrow-derived mesenchymal stem cells (hMSCs) with this biomaterial was examined. In addition, osteogenic differentiation was highly stimulated, which was confirmed by ALP (Alkaline phosphatase) activity and the ARS (Alizarin red S) staining of hMSCs. The pre-mixed-RCS@BGn satisfied the ISO standards for root canal sealers and maintained antimicrobial activity. Moreover, pre-mixed-RCS@BGn with more BGns turned out to have less cytotoxicity than pre-mixed-RCS without BGns while promoting osteogenic differentiation, mainly due to calcium and silicon ion release. Our results suggest that BGns enhance the biological properties of this calcium silicate-based sealer and that the newly introduced pre-mixed-RCS@BGn has the capability to be applied in dental procedures as a root canal sealer. Further studies focusing more on the biocompatibility of pre-mixed-RCS@BGn should be performed to investigate in vivo systems, including pulp tissue

Synergetic strengthening of layered steel sheet investigated using an in situ neutron diffraction tensile test

Synergetic strengthening induced by plastic strain incompatibility at the interface, and the resulting extra geometrically necessary dislocations (GNDs) generated during plastic deformation, were investigated to understand the origin of extra strength in heterogeneous structured (HS) materials. The mechanism of extra GND generation in twinning-induced plasticity (TWIP)-interstitial free (IF) steel layered sheet was quantitatively analyzed by conducting in situ neutron scattering tensile test. Load partitioning due to the different mechanical properties between the TWIP-steel core and IF-steel sheath at the TWIP/IF interface was observed during the in situ tensile testing. Because of the plastic strain incompatibility from load partitioning, extra GNDs are generated and saturate during tensile deformation. The extra GNDs can be correlated with the back-stress evolution of the HS materials, which contributes to the strength of layered materials. Because of the back-stress evolution caused by load partitioning, the strength of TWIP-IF layered steel is higher than the strength estimated by the rule-of-mixtures. This finding offers a mechanism by which extra GNDs are generated during load partitioning and shows how they contribute to the mechanical properties of HS materials.11Ysciescopu

Melatonin receptor 1 B polymorphisms associated with the risk of gestational diabetes mellitus

<p>Abstract</p> <p>Backgrounds</p> <p>Two SNPs in <it>melatonin receptor 1B </it>gene, <it>rs10830963 </it>and <it>rs1387153 </it>showed significant associations with fasting plasma glucose levels and the risk of Type 2 Diabetes Mellitus (T2DM) in previous studies. Since T2DM and gestational diabetes mellitus (GDM) share similar characteristics, we suspected that the two genetic polymorphisms in <it>MTNR1B </it>may be associated with GDM, and conducted association studies between the polymorphisms and the disease. Furthermore, we also examined genetic effects of the two polymorphisms with various diabetes-related phenotypes.</p> <p>Methods</p> <p>A total of 1,918 subjects (928 GDM patients and 990 controls) were used for the study. Two <it>MTNR1B </it>polymorphisms were genotyped using TaqMan assay. The allele distributions of SNPs were evaluated by <it>x</it>²models calculating odds ratios (ORs), 95% confidence intervals (CIs), and corresponding <it>P </it>values. Multiple regressions were used for association analyses of GDM-related traits. Finally, conditional analyses were also performed.</p> <p>Results</p> <p>We found significant associations between the two genetic variants and GDM, <it>rs10830963</it>, with a corrected <it>P </it>value of 0.0001, and <it>rs1387153</it>, with the corrected <it>P </it>value of 0.0008. In addition, we also found that the two SNPs were associated with various phenotypes such as homeostasis model assessment of beta-cell function and fasting glucose levels. Further conditional analyses results suggested that <it>rs10830963 </it>might be more likely functional in case/control analysis, although not clear in GDM-related phenotype analyses.</p> <p>Conclusion</p> <p>There have been studies that found associations between genetic variants of other genes and GDM, this is the first study that found significant associations between SNPs of <it>MTNR1B </it>and GDM. The genetic effects of two SNPs identified in this study would be helpful in understanding the insight of GDM and other diabetes-related disorders.</p

High resolution angle resolved photoemission studies on quasi-particle dynamics in graphite

We obtained the spectral function of the graphite H point using high resolution angle resolved photoelectron spectroscopy (ARPES). The extracted width of the spectral function (inverse of the photo-hole lifetime) near the H point is approximately proportional to the energy as expected from the linearly increasing density of states (DOS) near the Fermi energy. This is well accounted by our electron-phonon coupling theory considering the peculiar electronic DOS near the Fermi level. And we also investigated the temperature dependence of the peak widths both experimentally and theoretically. The upper bound for the electron-phonon coupling parameter is ~0.23, nearly the same value as previously reported at the K point. Our analysis of temperature dependent ARPES data at K shows that the energy of phonon mode of graphite has much higher energy scale than 125K which is dominant in electron-phonon coupling.Comment: 9 pages, 8 figures, accepted for publication in Phys. Rev.