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Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis. • Identification of transgene location is critical to assure monoclonality of CHO cells. • spPCR-based genome walking assay was developed and applied to recombinant CHO cells. • Python script-assisted selection of restriction enzyme can improve spPCR success rate. • spPCR saves required analysis time and cost substantially compared to current methods. • spPCR may aid cell line development by providing a simple and rapid analysis platform. [ABSTRACT FROM AUTHOR]

Sialylation, which is considered as a critical quality attribute, plays important roles in pharmacokinetics of recombinant proteins. Terminal sialic acids of glycoproteins are modulated by intracellular sialyltransferase (ST) and extracellular sialidase. Since CHO cells producing recombinant proteins can only transfer α2,3-sialic acid to a nascent oligosaccharide, inactivation of α2,6-ST results in a different composition of glycan with human proteins, which have predominantly α2,6-linkages. To overcome this problem and maximize total sialic acid contents, we used glucocorticoids (GCs) that are classified as a steroid hormone in CHO cells expressing both α2,3-ST and α2,6-ST. This study was performed to determine the effect of GCs on CHO cell cultures and sialylation of Fc-fusion protein. We observed that all cultures supplemented with GCs reduced the titer of the Fc-fusion protein, but enhanced the sialylation level, when compared to those of control. Especially, addition of corticosterone increases the levels of α2,6-sialylation as well as the total contents of sialic acid. Enhanced sialic acid contents are thought to be due to the intracellular improvement by sialylatransferase and the inhibition of sialidase. In conclusion, GCs can be utilized as an effective medium additive to improve the quality of biopharmaceuticals using CHO cell cultures. [ABSTRACT FROM AUTHOR]

A small molecular cage (4) with high affinity and complete selectivity for fluoride to the limit of detection over other competing small anions was synthesized. Cage 4 was also found to retain the encapsulated fluoride anion within its cavity even after one or two pyrrolic NH protons were subject to deprotonation. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the efficacy of combined grape pomace and omija fruit ethanol extracts (GO) on metabolic disorders in overweight or obese subjects. Seventy-six subjects (30-70 years, body mass index ≥23.0 kg/m2) were divided into control (starch, 4 g/day, n = 24), low-GO (low dose GO, grape pomace extract [342.5 mg/day] + omija fruit extract [57.5 mg/day], n = 26), and high-GO (high dose GO, grape pomace extract [685 mg/day] + omija fruit extract [115 mg/day], n = 26) groups. Body composition, nutrient intake, plasma lipid profiles, inflammation, antioxidant capacity, and hepatotoxicity markers were assessed in all subjects at the baseline and 10 weeks after taking the supplements. The body weight and body fat of overweight or obese subjects was not significantly altered in the low-GO and high-GO groups. However, the high-GO supplement significantly decreased the baseline-adjusted final plasma total-cholesterol, low-density lipoprotein (LDL)-cholesterol, and non-high-density lipoprotein (HDL)-cholesterol levels and increased the baseline-adjusted final plasma apolipoprotein (apo) A-1 level compared with that of the control group. In addition, the high-GO supplement significantly lowered apo B, apo B/apo A-1, lipoprotein a (Lp[a]), atherogenic index, interleukin (IL)-1 β, tumor necrosis factor- α, and elevated erythrocyte antioxidant capacity compared with the control group or the baseline levels. The low-GO supplement decreased the plasma IL-1 β level and elevated erythrocyte superoxide dismutase activity compared with that at baseline. However, in general, high-GO exerted a greater effect than low-GO. There were no significant differences in activities of plasma glutamate oxaloacetate transaminase and glutamate pyruvate transaminase between the groups. This study is a preliminary clinical study to verify that GO could be beneficial for amelioration of obesity-related dyslipidemia, inflammation, and oxidative stress without side effect in the overweight or obese subjects. [ABSTRACT FROM AUTHOR]

Transparent conducting films (TCFs) were fabricated using single-wall carbon nanotube (SWCNT)–silver nanowire (AgNW) hybrid materials on flexible substrates. The hybrid solution was prepared with various weight ratios of SWCNTs and AgNWs, followed by bar-coating on polyethylene terephthalate (PET) film. As the SWCNT content increased, the haze of the film decreased markedly, while transparency and sheet resistance were a little bit impaired. As a result, the prepared hybrid TCFs exhibited a sheet resistance of 120 Ω/sq and a transparency of 90.8%, with a haze of as low as 1.6% when the weight ratio of SWCNTs to AgNWs was 2:3. These properties appear suitable for the application of TCFs in high-performance, flexible capacitive touch panels. [ABSTRACT FROM AUTHOR]

The stacked CuGa/In metallic precursor thin films were fabricated and annealed at low temperature of 200°C before the selenization for fabricating Cu(In,Ga)Se2photovoltaic absorber. The pre-annealing of Cu-In-Ga precursor was found to reduce Cu9Ga4phase and increase the formation of Cu16(In,Ga)9phase, which may be preferable for the subsequent selenization in terms of homogenizing Ga across the absorber thickness. However, the replacement of In with Ga in the ternary phase produced large amount of free-In, which gave rise to blistered area as shunt paths after the selenization. The performance of solar cell fabricated using the pre-annealed precursor was heavily deteriorated compared to the not-annealed precursor. [ABSTRACT FROM AUTHOR]

We successfully fabricated the TiO2-based photoelectrode doped with reduced graphene oxide (rGO) to improve the power conversion efficiency of dye-sensitized solar cells (DSSCs). The ratio of rGO was 0.3∼1.5 wt%. The DSSCs with the TiO2photoanode incorporated 0.3 wt% rGO showed a maximum power conversion efficiency of 6.26% which is 44% higher than the pristine TiO2photoelectrode. We concluded the introduction of rGO doped at 0.3 wt% ratio contributed to reducing the resistance of the surface and the interface of the photoelectrode from the observation of electrochemical impedance spectroscopy(EIS). [ABSTRACT FROM AUTHOR]

Plasmodium vivax merozoite surface protein 1 (PvMSP1) is believed to be important in erythrocyte invasion. However, the detailed mechanism of PvMSP1-mediated invasion has been unclear. We demonstrate that the C-terminal 19 kDa domain (PvMSP119) of PvMSP1, the 42-kDa fragment of PvMSP1 is further cleaved to a 33 kDa N-terminal polypeptide and a 19 kDa C-terminal fragment in a secondary processing step, is a critical domain in the binding between parasite ligand and erythrocyte receptor. Also, its cytoadherence was successfully blocked by naturally acquired immunity, was partially sensitive to neuraminidase and trypsin. When expressed separately epidermal growth factor (EGF)-like motifs 1 and 2, subunits of the PvMSP119, mediated 64% and 66% of the erythrocyte-binding activity, respectively, relative to their expression together as a single intact ligand domain. These results suggest that the EGF-like motifs 1 and 2 of PvMSP119 function as a core-binding portion in the attachment of PvMSP1 to erythrocytes. [Copyright &y& Elsevier]

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes. [ABSTRACT FROM AUTHOR]

Simple Summary: This study explores the use of the 2020 ESGO/ESTRO/ESP molecular classification system in predicting survival and recurrence rates in patients with endometrial cancer. It aims to assess the effectiveness of this new classification by comparing it with previous ESMO guidelines. By incorporating both clinicopathological and molecular factors into the risk assessment, this research seeks to offer a more precise method for categorizing patients, potentially leading to more tailored and effective treatment strategies. The findings could significantly influence future approaches to managing endometrial cancer, emphasizing the importance of molecular risk categorization in improving patient outcomes. This study aimed to evaluate the efficacy of the 2020 European Society of Gynecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) guidelines for endometrial cancer (EC). Additionally, a novel risk category incorporating clinicopathological and molecular factors was introduced. The predictive value of this new category for recurrence and survival in Korean patients with EC was assessed, and comparisons were made with the 2013 and 2016 European Society of Medical Oncology (ESMO) risk classifications. Patients with EC were categorized into the POLE-mutated (POLEmut), mismatch repair-deficient (MMRd), p53-aberrant (P53abn), and nonspecific molecular profile (NSMP) subtypes. Recurrence, survival, and adjuvant therapy were assessed according to each classification. Notably, patients with the POLEmut subtype showed no relapse, while patients with the P53abn subtype exhibited higher recurrence (31.8%) and mortality rates (31.8%). Regarding adjuvant therapy, 33.3% of low-risk patients were overtreated according to the 2020 ESGO/ESTRO/ESP guidelines. Overall and progression-free survival differed significantly across molecular classifications, with the POLEmut subtype showing the best and the P53abn subtype showing the worst outcomes. The 2020 ESGO molecular classification system demonstrated practical utility and significantly influenced survival outcomes. Immunohistochemistry for TP53 and MMR, along with POLE sequencing, facilitated substantial patient reclassification, underscoring the clinical relevance of molecular risk categories in EC management. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cell cultures are widely used due to their high productivity, industrial track record, safety record, and correct post-translational modifications. Fed-batch and perfusion modes have been mostly adopted as traditional commercial manufacturing process using CHO cells for the production of biopharmaceuticals. Thus, we compared fed-batch and perfusion culture processes for the commercial-scale production of monoclonal antibody. Main production process was performed after confirming the comparable culture performance in seed train of different operation modes, and then perfusion cultures showed 1.6-fold higher peak viable cell density (VCD) with a longer culture duration than that of fed-batch cultures. Also, as the result of main batches, total product amount increased over 450% in perfusion cultures. Importantly, perfusion process allowed to improve the higher purification yield while maintaining acceptable product quality, because the level of process impurity could be reduced by using retention device. When economic comparison of fed-batch versus perfusion processes was carried out under the equal scale of upstream and downstream, perfusion mode was more a cost-effective bioprocess that could reduce the cost of goods. In conclusion, although comparison data of triplicate commercial batches are of course specific case for a given antibody production, this study verified the possibility of using perfusion mode as an efficient system for commercial producing the desired antibody and is a meaningful report in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N -acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (−10.9% and −18.6%, respectively), while bi- and tri-sialylated N -glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N -glycans decreased from 32.0% to 28.5%, while that of sialylated N -glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines. • Protein-specific glycoengineered CHO cells were constructed. • Overexpression of GnT-IV played a major role in antennary structure formation of CTLA4-Ig. • Co-overexpression of GnT-IV and α2,6-ST made highly sialylated CTLA4-Ig. [ABSTRACT FROM AUTHOR]

The photovoltaic performances of i-ZnO/CdS/Cu(In,Ga)Se2(CIGS) solar cell with different window architectures of SnO2:In2O3(ITO), Al2O3:ZnO (AZO) and ITO/AZO, were experimentally compared. The solar cell with ITO deposited directly on i-ZnO layer showed an abnormal current-voltage characteristic as having a shunt path. Both of AZO and ITO/AZO resulted in normal current-voltage behavior as far as AZO is contacting ZnO. [ABSTRACT FROM AUTHOR]

The Cu(In,Ga)Se2(CIGS) thin film solar cells were fabricated using ZnS buffer layer deposited by chemical bath deposition and ZnO/Al-doped ZnO transparent conducting layers deposited by magnetron sputtering with two different configurations of target-to-substrate angle and distance in order to evaluate the effect of plasma damage on device performance. The higher efficiency was obtained from the configuration of the substrate being far away from the target center than face-to-face configuration, suggesting that the excessive bombardment of high energy particles from the plasma on the film surface is responsible for the solar cell performance degradation. It is suggested that the improved open-circuit voltage is ascribed to the increased recombination barrier height. [ABSTRACT FROM AUTHOR]

In this study, in an effort to increase the surface Ga contents of Cu(In,Ga)Se2absorber, a high Ga-containing sputter target (Cu-30 at%Ga) was used to produce a precursor and was compared with Cu-22 at%Ga target in terms of photovoltaic performances of completed solar cells. The precursors consisted of multi-stack of CuGa/In and were selenized by using Se vapor at 530°C. The higher efficiency was obtained from the high Ga target with substantial improvement of open-circuit voltage and short-circuit current density. The increased hole concentration with more Ga addition in the absorber was found to be responsible for the higher open-circuit voltage. [ABSTRACT FROM AUTHOR]

Cu(In,Ga)Se2(CIGS) films were deposited by the three-stage co-evaporation process and the effect of the second stage substrate temperature on device performance were evaluated. With increasing the substrate temperature, the preferred orientation of CIGS was found to be changed from (112) to (220), however, the Ga double grading became weaker and nearly flat for the (220) textured CIGS grown at 570°C. Despite of (220) preferred orientation, the conversion efficiency decreased mainly due to the poor fill factor and open circuit voltage. It is suggested that increasing the growth temperature in order to get (220) preferred CIGS may destroy the advantage of double-graded Ga profile. [ABSTRACT FROM AUTHOR]