Cho and Pak reply to Lamm et al. comment on "A Convergent Series for the QED Effective Action"

Cho and Pak reply to Lamm et al. [hep-th/0007108] comment on "A Convergent Series for the Effective Action of QED" [hep-th/0006057].Comment: 1 pag

The effect of a carbohydrate mouth rinse on performance of the yo-yo intermittent recovery level 1 test with female university level hockey players

It has been suggested that carbohydrate mouth rinse (CHO-MR) effects short duration, high intensity exercise by activation of sensory receptors on the tongue (Carter et al., 2004: Medicine and Science in Sports and Exercise, 36, 2107–2111). Research has predominately focused on the effects of CHO-MR on the performance of cycling and running time trials (Painelli et al., 2010: Nutritional Journal, 9, 1-4). Limited research has been conducted on the effectiveness of a CHO-MR on intermittent high-intensity field-based sports performance. The aim of this study was to analyse the effects of a CHO mouth rinse on performance of the Yo-yo Intermittent Recovery Level 1 Test (IR1T) with female University standard hockey players. Following ethical approval, twelve members (mean age 20 ± 0.98 years, stature 167 ± 7.09 cm, and body mass 64.7 ± 4.96 kg) of the University of Lincoln’s female 1st team volunteered for the study. The study used a single-blind counter-balanced design with repeated measures on two treatment conditions: 1) a CHO-MR, and 2) a placebo mouth rinse (PL-MR). Participants were instructed to maintain a normal diet and fasted for 12hr prior to testing. After a familiarisation test, twelve participants completed the IR1T twice, one week apart; rinsing with either a CHO-MR or PL-MR. Administration occurred before (20min prior) the IR1T and during the 10s active recovery periods, at intervals (IV) which corresponded to five level increments in speed (IV1-13.5km/h, IV2-14km/h, IV3-14.5km/h, IV4-15km/h, IV5-15km/h). At these points, rate of perceived exertion (RPE) was recorded using the traditional Borg scale. Total distance (m) achieved was recorded as the performance measure. A dependent t-test did not detect any performance improvement (P = >0.05) between CHO-MR (1060 ± 273m) and PL-MR (1127 ± 402m) trials. Multiple dependent t-tests revealed that at the first IV (SL 12.1), RPE scores were significantly different (P = 0.006) between CHO-MR (10.9 ± 0.79) and PL-MR (11.4 ± 1.08) trials. No differences were detected between CHO-MR and PL-MR trials during the rest of the protocol (IV2-5, all P = >0.05). A CHO-MR had no effect on IR1T test performance with female university level hockey players compared to a PL-MR. The participants did not experience any differences in the feeling of exertion between the two conditions as the IR1T progressed. Further research needs to illuminate any possible performance effects from CHO-MR with intermittent high intensity activity, revealing any plausible physiological mechanisms of action

The Effects of Carbohydrate, Protein, and Carbohydrate with Protein Solutions on 200-Meter Sprint Speed

Purpose: To investigate the differential effects of solutions providing varying concentrations of carbohydrate and/or protein ingested between 200-meter sprints on sprint time. Subjects: Recruitment was from the Georgia State University track and field team. Methods: The study protocol was approved by the Georgia State University IRB. Ten subjects, 18 to 21 years of age, consented to be included in the study. Nine subjects (7 females; 2 males) completed trial 1, six subjects (5 females; 1 male) completed trial 2, and three subjects (2 females; 1 male) completed the final trial. Each trial consisted of a 200-meter sprint followed by the immediate ingestion of a post-exercise recovery beverage within the first fifteen minutes of a one-hour recovery period. Following the one-hour of recovery, subjects sprinted a second 200-meter sprint. Beverage solutions were formulated to contain 1.2 g of protein (PRO), 1.2 g carbohydrate (CHO), or 1.2 g carbohydrate with protein (CHO/PRO) per kg of subject body weight. Using a single blind, non-randomized design, subjects received the same recovery beverage in each trial. Each trial consisted of either PRO (trial 1), CHO (trial 2), or CHO/PRO (trial 3), with one week separating trials. Sprint times were recorded in seconds and ten hundredths of a second using a manual, digital stopwatch. Results: During PRO, two subjects sprinted faster (x= -.25 sec), three subjects saw no change in sprint time, and four subjects sprinted slower (x= +.98 sec). During CHO, two female subjects sprinted faster between sprints (x= -.85 sec); and all other subjects (n=4) sprinted slower (x= +.73 sec). During CHO/PRO, no subjects sprinted faster from sprint 1 to sprint 2 (x= +.33 sec) Conclusions: Post-exercise nutritional supplementation effects varied among subjects, with some subjects performing better following PRO, while others experiencing improvements with CHO. In general, subjects performed better following consumption of the CHO beverage. Of those who ran faster between sprints, the CHO beverage resulted in an average improvement of -.85 sec, while the PRO beverage resulted in an average improvement of -.25 sec. On average, CHO resulted in faster 2nd sprints (x= +.20 sec) than the PRO beverage (x= +.47 sec) or the CHO/PRO beverage (x= +.33 sec). Continued research in this population is necessary for elucidation of study results. This investigation may serve as the foundation for future, related studies

High-yield antibody production using targeted integration and engineering CHO host

To identify the high expression sites in the CHO cells, we employed NGS to analyze the integration sites of a high producing cell line (titer \u3e 3g/L). The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. To test the expression activity of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed 4 integration sites are in the high producing cell line. Among the 4 integration site, one integration site was tested by CRISPR/Cas9 for target integration of antibody gene for expression. The target integrated cell pool present higher expression level (130 mg/L/copy) and less copy number when compared other integration sites. Through single-copy integration method, we can also achieve 60-150 mg/L/copy in a batch culture. About 80% of the single-copy cell clones were stable at generation 60. We have also applied the CHO-specific microarray transcriptomics technology to identify genes that contribute to high productivity. Transfection of our proprietary dual promoter vector J 1.0 resulting in 1.65 to 2.4 fold increase in the expression in engineered CHO DXB11 host. Through fed-batch process development, 3 – 5 g/L mAb productivity can be achieved through targeted integration and engineered CHO host

CHO-K1 host cell engineering strategy enabling the establishment of strains producing higher yields of recycling antibodies

As described elsewhere (Biotechnol Bioeng 2010, 2013), our DXB11 (dhfr–) host cell engineering strategy achieved high cell viability for a prolonged period (more than 1 month) and enhanced mAb productivity (\u3e100 pg/cell/day) by nutritional control. Introduction of taurine transporter (TAUT) into DXB11 parent cells increased glutamine uptake and accelerated glutathione metabolism. By forcing the overexpression of TAUT, we were able to control DXB11 host cell functions and thereby increase the monoclonal antibody (mAb) titer up to 8.1 g/L/31 days under conventional 1-L bioreactor fed-batch conditions. Furthermore, the mAb produced by the DXB11/TAUT cells was comparable in quality to the mAb produced by the parent cells. In this study, we used CHO-K1 host cells and a chemically defined medium (CDM) for the development of cell lines producing recycling antibodies (rcAb). CDM adaptation and TAUT overexpression improved CHO-K1 cell performance. Rapid-growth CHO-K1/TAUT cells were developed, and these enabled the establishment of strains that produced higher yields of rcAb than did CHO-K1 parent cell (p \u3c 0.05). Viable cell density of these CHO-K1/TAUT/rcAb strains increased not only under shaker passage culture conditions (p \u3c 0.01) but also under shaker fed-batch culture conditions (p \u3c 0.01). These results suggest that our TAUT overexpression strategy also has a unique potential for the improvement of CHO-K1 host cells as well as DXB11 host cells

High titer transient gene expression platform based on GS CHO cell line – rapid protein expression tool for preclinical drug development

Most of the high yielding transient gene expression (TGE) methods for CHO cells reported in the literature involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. However, this is a laborious, time intensive process and also requires specific vector engineering for transient expression. Here, we present results from development of a high titer TGE process based on GS-CHO cells without resorting to host cell line engineering or TGE specific vector engineering. This was achieved by optimization of direct addition of DNA and PEI, use of DMA to enhance transgene mRNA levels and addition of a proprietary feeds. Protein titers of up to 0.35 g/L or 1 g/L were reached in a 7 day or a 16 day bioprocess respectively. Robustness and reproducibility of this method was demonstrated at scales ranging from 2 ml to 2 L. Moreover, these transfections could be performed in a high throughput manner in 24 deep well plates. Taking advantage of this, we coupled this high throughput transfection process to a high-throughput semi-automated Protein A purification process capable of purifying up to 72 unique mAbs simultaneously. We designed a variable volume elution strategy based on supernatant titer and were able to obtain protein yields of 0.25 mg to 1 mg at concentration \u3e0.5 mg/ml. In efforts to further increase protein expression levels, we tested co-transfection of proteins involved in the unfolded protein response with the goal to enhance secretion capabilities of the transfected cell population. We identified that co-transfection of plasmid DNA encoding XBP1S increased protein expression by 15% to 80%. We also present case studies comparing product quality attributes from transient HEK293, transient CHO and stable CHO pool. Overall, our results demonstrate that a high yielding and representative transient CHO process can be developed while maintaining the speed and simplicity of a TGE process

In vivo action of RNA G-quadruplex in phloem development

Phloem network integrates cellular energy status into post-embryonic growth, and development by tight regulation of carbon allocation. Phloem development involves complicated coordination of cell fate determination, cell division, and terminal differentiation into sieve elements (SEs), functional conduit All of these processes must be tightly coordinated, for optimization of systemic connection between source supplies and sink demands throughout plant life cycle, that has substantial impact on crop productivity. Despite its pivotal role, surprisingly, regulatory mechanisms underlying phloem development have just begun to be explored, and we recently identified a novel translational regulatory network involving RNA G-quadruplex and a zinc-finger protein, JULGI, for phloem development From this perspective, we further discuss the role of RNA G-quadruplex on post-transcriptional control of phloem regulators, as a potential interface integrating spatial information for asymmetric cell division, and phloem development.11Ysciescopuskc