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

Real-time response estimation of structural vibration with inverse force identification

This study aimed to develop a virtual sensing algorithm of structural vibration for the real-time identification of unmeasured information. First, certain local point vibration responses (such as displacement and acceleration) are measured using physical sensors, and the data sets are extended using a numerical model to cover the unmeasured quantities through the entire spatial domain in the real-time computation process. A modified time integrator is then proposed to synchronize the physical sensors and the numerical model using inverse dynamics. In particular, an efficient inverse force identification method is derived using implicit time integration. The second-order ordinary differential formulation and its projection-based reduced-order modeling is used to avoid two times larger degrees of freedom within the state space form. Then, the Tikhonov regularization noise-filtering algorithm is employed instead of Kalman filtering. The performance of the proposed method is investigated on both numerical and experimental testbeds under sinusoidal and random excitation loading conditions. In the experimental test, the algorithm is implemented on a single-board computer, including inverse force identification and unmeasured response prediction. The results show that the virtual sensing algorithm can accurately identify unmeasured information, forces, and displacements throughout the vibration model in real time in a very limited computing environment.Comment: 24 Pages, 15 Figures, 10 Table

CRISPR RNAs trigger innate immune responses in human cells

Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to similar to 80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting St-hydroxyl gRNAs in complex with Cas9 or Cpfl avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4(+) T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

Evaluation of the physicochemical, metabolomic, and sensory characteristics of Chikso and Hanwoo beef during wet aging

Objective This study aimed to evaluate the physicochemical, metabolomic, and sensory qualities of Chikso and Hanwoo beef during 28 days of wet aging. Methods Rump and loins from Hanwoo and Chikso were obtained and wet-aged for 28 days at 4°C. The samples were collected at 7-day interval (n = 3 for each period). Physicochemical qualities including pH, meat color, shear force value, and myofibrillar fragmentation index, metabolomic profiles, and sensory attributes (volatile organic compounds and relative taste intensities) were measured. Results Chikso showed a significantly higher shear force value than Hanwoo on day 0; however, no differences between breeds were found after day 14, regardless of the cuts. Overall, Chikso had more abundant metabolites than Hanwoo, especially L-carnitine and tyrosine. Among the volatiles, the ketone ratio was higher in the Chikso rump than the Hanwoo rump; however, Chikso had fewer alcohols and aldehydes than Hanwoo. Chikso rump showed higher taste intensities than the Hanwoo rump on day 0, and sourness decreased in Chikso, but increased in the Hanwoo rump on day 14. Wet aging for 14 days intensified the taste of Chikso loin but reduced the umami intensity of Hanwoo loin. Conclusion Chikso had different metabolomic and sensory characteristics compared to Hanwoo cattle, and 14 days of wet aging could improve its tenderness and flavor traits

Approaches for Sample Characterization and Lithography with Nanoparticles using Modes of Scanning Probe Microscopy

Measurement and imaging modes of scanning probe microscopy (SPM) have been routinely applied for characterizing systems of nanoparticles; however the evolution of fabrication methods to prepare arrangements of nanoparticles remains a challenge. Reproducible fabrication of surface structures which integrate nanoparticles within ultra-small patterns will require innovative approaches to achieve high throughput and precision. Strategies for nanoscale lithography have been introduced for preparing defined arrangements of nanoparticles on surfaces based on physical or chemical interactions. For example, physisorption was employed for attaching nanoparticles based on colloidal lithography and site-directed assembly. Microfabricated atomic force microscope (AFM) tips with capillary channels have been used to pattern nanoparticles through electrostatic interactions. Specific chemical interactions can be designed for patterning nanoparticles with dip-pen nanolithography and SPM-based fabrication. Studies with nanoparticles are reviewed, which have applied either in situ and ex situ approaches for imaging and measurements using modes of SPM. The imaging principle for contact and tapping modes are described with example studies of nanoparticle patterns. The SPM modes for measuring physical properties (e.g. magnetism, softness, conductance) using force modulation microscopy (FMM), magnetic force microscopy (MFM), magnetic sample modulation (MSM), and conductive probe AFM are described for selected studies of lithography with nanoparticles. Strategies for patterning nanoparticles using lithography modes of nanoshaving, dip-pen nanolithography, and tip-induced oxidation have been reported for a range of nanoparticle systems. Applications for nanotechnology will require the integration of nanoparticles within engineered surface architectures. Stable, organized arrangements of nanoparticles with robust chemical/physical attachment to surfaces will be needed for applications, to fully gain advantages of the characteristic quantum properties of nanoparticles