Genomic understanding of clonal variation in recombinant CHO cells

In the current manufacturing platform for large-scale production of therapeutic proteins, recombinant Chinese hamster ovary (rCHO) cell line generation is probably the most time-consuming step. The high degree of phenotypic heterogeneity in the absolute transgene expression level and variable responses to culture conditions among the selected clones, which has been referred to as “clonal variation”, makes the cell line generation process laborious and time-consuming. Upon transfection, a transgene is randomly integrated into the chromosomes of the host cells, and the integration site of the transgene is believed to be responsible for this clonal variation. However, the lack of understanding the molecular basis involved in clonal variation has hindered rapid cell line generation with a predicted culture performance. With the availability of CHO genome sequences and targeted genome sequencing/editing technologies, we investigated “clonal variation” with an emphasis on the transgene integration sites and configuration of the integrated transgenes. rCHO cell clones expressing GFP, which were constructed by random integration of the GFP expression vector into CHOK1 host cells, revealed a remarkable variability in GFP expression at 37°C. Furthermore, they showed different responses to culture temperature shifts from 37°C to 33°C confirming the existence of clonal variation. Application of targeted sequencing by the proximity ligation to the transgenes enabled the mapping of the integrated transgenes, and thereby the genomic integration sites of the transgene in the representative rCHO cell clones showing different responses to hypothermia were identified. To determine whether the different responses of the rCHO clones to hypothermia were due to the different integration sites of the transgenes, rCHO cell clones expressing GFP were also constructed by CRISPR/Cas9-mediated targeted integration of an intact transgene into CHOK1 host cells at the integration sites that were identified in the rCHO cells exhibiting different responses to hypothermia. Surprisingly, the rCHO clones constructed with targeted integration, regardless of the integration site of the gene, had similar expression patterns in terms of the absolute expression level and responses to hypothermia. They all exhibited enhanced GFP expression with hypothermia. Evidence of several rearrangements in the integrated transgene was detected in the rCHO clones constructed with random integration, which may interrupt the normal function of regulatory elements, particularly promoters. Promoter replacement and dissection results support the crucial role of promoter elements in the differential transgene expression patterns at the identical genomic site. Taken together, we demonstrate the complex nature of “clonal variation” in rCHO cells which encompasses the concept of vector elements and their rearrangement upon random integration besides just the genomic integration sites. Further characterization of the interactions between the integration sites and vector regulatory elements together with controlled integration of transgenes could lead to the tailored control of recombinant gene expression in rCHO cells while minimizing clonal variation

Radicalization of Airspace Security: Prospects and Botheration of Drone Defense System Technology

The development of a comprehensive and decisive drone defense integrated control system that can provide maximum security is crucial for maintaining territorial integrity and accelerating smart aerial mobility to sustain the emerging drone transportation system (DTS) for priority-based logistics and mobile communication. This study explores recent developments in the design of robust drone defense control systems that can observe and respond not only to drone attacks inside and outside a facility but also to equipment data such as CCTV security control on the ground and security sensors in the facility at a glance. Also, it considered DDS strategies, schema, and innovative security setups in different regions. Finally, open research issues in DDs designs are discussed, and useful recommendations are provided. Effective means for drone source authentication, delivery package verification, operator authorization, and dynamic scenario-specific engagement are solicited for comprehensive DDS design for maximum security Received: 2023-03-07 Revised: 2023-04-2

Accelerated homology-directed targeted integration of transgenes in CHO cells via CRISPR/Cas9 and fluorescent enrichment

Development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. With draft genome of several CHO cell lines and targeted genome editing technologies, rCHO cell line development based on site-specific integration has the potential to overcome the limitations of clonal heterogeneity. In a previous study, we demonstrated efficient and precise targeted gene integration in CHO cells using CRISPR/Cas9 technology and homology-directed repair (HDR) pathway1). However, it requires a selection process, which limits targeted integration of multiple transgenes at multiple sites due to a limited number of selection markers and a lengthy selection process. Here, we improved the targeted integration platform by applying fluorescent enrichment of transfected cells. The improved system is based on a fluorescent protein A linked Cas9 together with sgRNA towards the integration site and donor DNA harboring a fluorescent gene B outside homology regions. Involvement of fluorescent markers in constructs confers FACS enrichment of cells transfected with both Cas9 and donor DNA. Simultaneous introduction of GFP 2A peptide-linked Cas9 and sgRNA expression vectors together with donor plasmid has enabled precise targeted integration of large transgenes encoding model proteins including antibody, following transient expression and FACS enrichment. Subsequent selection of non-fluorescent clonal cells allowed for excluding cells with randomly integrated donor DNA. In this way, we not only shortened the clone screening time, but also generated clonal CHO cell lines with site-specific, marker-free (no antibiotic selection needed), and clean (no unwanted DNA present) targeted integration of GOI. Further improvement in targeted integration efficiency was additionally assessed by chemical treatment toward cell cycle arrest or nonhomologous end joining inhibition combined with fluorescent enrichment. Taken together, the present platform has the huge potential to accelerate targeted generation of stable production CHO cell lines in a rational way

Changes of phenolic compounds in LebZIP2-overexpressing transgenic plants

484-491The bZIP gene is a transcription factor that plays various roles in relation to plant stress and hormone signaling. This gene is also involved in plant environmental stress and herbicide tolerance. We generated Nicotiana benthamiana transgenic plants with LebZIP2-encoding gene isolated from tomatoes using Agrobacterium-mediated transformation. Transgenic seeds harvested from these T0 transgenic plants were grown and examined for gene transfer and changes in phenolic compounds in the T1 generation. RT-PCR analysis using a primer specific to the LebZIP gene confirmed that the gene was transferred to the T1 generation. We analyzed the increase and decrease tendency for 30 phenolic compounds using the T1 generation-transgenic plants and investigated the mechanism between the specifically increased compound and LebZIP2 gene. Gallic acid, homogentisic acid, protocatechuic acid, myricetin, t-cinnamic acid, and b-resorcyclic acid were identified as the phenolic compounds that increased in T1 transgenic plants overexpressing the LebZIP gene. Among these, homogentisic acid at 246.75-1055.19 µg/g, was increased by 2-5 fold in the T1 transgenic plants compared to the control. Protocatechuic acid was found at 1640.54-2456.00 µg/g and was increased by 2-4 fold in T1 transgenic plants. t-Cinnamic acid was present in a small amount of 23.14 µg/g in the control, whereas it was 102.19-135.47 µg/g in T1 transgenic plants, showing an increase of 4-5 folds. These results indicated that homogentisic acid, protocatechuic acid, and t-cinnamic acid among the 30 phenolic compounds analyzed, were significantly increased in LebZIP2-overexpressing T1 transgenic plants, and support the evidence that the LebZIP2 gene is significantly involved in the increment of three phenolic compounds

Trends and Associated Factors of Use of Opioid, Heroin, and Cannabis Among Patients for Emergency Department Visits in Nevada: 2009–2017

To examine trends and contributing factors of opioid, heroin, and cannabis-associated emergency department (ED) visits in Nevada. The 2009 to 2017 Nevada State ED database (n = 7,950,554 ED visits) were used. Use of opioid, heroin, and cannabis, respectively, was identified by the International Classification of Diseases, 9th & 10th Revisions. Three multivariable models, one for each of the 3 dependent variables, were conducted. Independent variables included year, insurance status, race/ethnicity, use of other substance, and mental health conditions. The number of individuals with opioid, heroin, cannabis-associated ED visits increased 3%, 10%, and 23% annually from 2009 to 2015, particularly among 21 to 29 age group, females, and African Americans. Use of other substance (odds ratio [OR] = 3.91; 95% confidence interval [CI] = 3.84, 3.99; reference - no use of other substance), mental health conditions (OR = 2.48; 95% CI = 2.43, 2.53; reference – without mental health conditions), Medicaid (OR = 1.41; 95% CI = 1.38, 1.44; reference – non-Medicaid), Medicare (OR = 1.44; 95% CI = 1.39, 1.49; reference – non-Medicare) and uninsured patients (OR = 1.52; 95% CI = 1.49, 1.56; reference - insured) were predictors of all three substance-associated ED visits. With a steady increase in trends of opioid, heroin, and cannabis-associated ED visits in recent years, the main contributing factors include patient sociodemographic factors, mental health conditions, and use of other substances

Performance Analysis On A Variable Capacity Swash Plate Compressor

A numerical study on the performance of a variable capacity swash plate compressor for an automotive air-conditioning system was carried out. The compressor under investigation had six cylinders and capacity regulation was made by changing the swash plate inclination angle. A numerical simulation program was made based on mathematical modelings on the swash plate dynamics, refrigerant states in various control volumes such as cylinders and crank room, and flows in the opening passages of electric control valve for crank room pressure control. The simulation results such as mass flow rate, compressor power consumption, cooling capacity and COP were compared with measurements within ±5% deviation over various operating conditions except at low operating speed such as idling condition. By using the simulation program, the effect of the crank room pressure on the swash plate inclination angle and the determination of the crank room pressure level by the electric control valve openings could be investigated

Hybrid cell line development system utilizing site-specific integration and methotrexate- mediated gene amplification in Chinese Hamster Ovary cells

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Flake Orientation in Injection Molding of Pigmented Thermoplastics

In the present work, experimental studies are carried out to understand orientation kinematics of pigment flakes during the injection molding process. The injection molding experiments are carried out using ABS resin compounded with aluminum flakes. Thin specimens are sliced off from the injection molded sample, and then the orientation distribution is observed using transmitted microscopy. Generally, the microscopic result shows a sandwich structure where the orientation state near the mid-plane differs significantly from that around the surface. Particularly at the weldline region, locally different orientation is observed near the part surface, which is the result of fountain flow at the melt front. Also the effect of mold temperature on the flake orientation is presented

Nitrogen Budget of South Korea from 2015 to 2019 Including Gaseous Nitrogen Oxides

Objectives The study was aimed to systematically understand the nitrogen budget from 2015 to 2019, and to determine the annual nitrogen balance in South Korea. Methods The nitrogen budget was calculated by classifying into agricultural and livestock, city, and forest areas. The nitrogen input was estimated from deposition, biological fixation, agricultural water, inorganic chemical fertilizer, compost, feedstuff, produced crops, imported grocery, and fuel consumption. The nitrogen output was estimated by considering denitrification, volatilization, ground water runoff, land and crop absorption, sewage disposal, drainage, forest uptake, and nitrogen oxide (NOx) emissions. Results and Discussion The annual nitrogen budget from 2015 to 2019 showed the input as 6,107,449 ton/yr, 6,144,666 ton/yr, 6,245,240 ton/yr, 6,281,552 ton/yr, 5,993,347 ton/yr and the output as 1,346,587 ton/yr, 1,369,119 ton/yr, 1,365,327 ton/yr, 1,378,940 ton/yr, 1,365,994 ton/yr, respectively. The NOx, highly contributing to change in the nitrogen budget, was caused by human activities such as vehicles, business places, and power plants. More than 32% of the total nitrogen budget was caused by NOx in every year. Conclusion The nitrogen budget continued to increase in the 2010s because of the increase in food and fuel consumption by urbanization. However, the nitrogen budget declined in 2019 due to the effort such as emission regulations and the use of alternatives as the awareness of environmental pollution gradually increased. Therefore, the appropriate institution should be established and actively implemented to reduce the nitrogen effectively