Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Non-Newtonian Dynamics in Water-in-Salt Electrolytes.

Water-in-salt electrolytes have attracted considerable interest in the past decade for advanced lithium-ion batteries, possessing important advantages over the non-aqueous electrolytes currently in use. A battery with a LiTFSI-water electrolyte was demonstrated in which an operating window of 3 V is made possible by a solid-electrolyte interface. Viscosity is an important property for such electrolytes, because high viscosity is normally associated with low ionic conductivity. Here, we investigate shear and longitudinal viscosities using shear stress and compressional longitudinal stress measurements as functions of frequency and concentration. We find that both viscosities are frequency-dependent and exhibit almost identical frequency and concentration dependences in the high-concentration region. A comparison to quasielastic neutron scattering experiments suggests that both are governed by structural relaxation of the TFSI- network. Thus, LiFTSI-water electrolytes appear to be an unusual case of a non-Newtonian fluid, where shear and longitudinal viscosities are determined by the same relaxation mechanism.

Coexistence of two coacervate phases of polyglycine in water suggested by polymer reference interaction site model theory.

Mixing Gibbs energy and phase equilibria of aqueous solutions of polyglycine were studied theoretically by means of polymer reference interaction site model integral equation theory combined with the Gibbs-Duhem method. In addition to the ordinary liquid-liquid phase separation between dilute and concentrated solutions, the theoretical calculation predicted the coexistence of two coacervate phases, namely, the lower- and higher-density coacervates. The relative thermodynamic stabilities of these two phases change with the polymerization degree of polyglycine. The higher-density coacervate phase was rapidly stabilized by increasing the polymer length, and the lower-density phase became metastable at large polymers. The hydrogen bonds between the peptide chains were strengthened, and water was thermodynamically destabilized in the higher-density coacervate. A possible relation with the formation of amyloid fibril within a liquid droplet is also discussed.

Knowing the learning strategy is not enough to use it: Example in reading strategies for Japanese undergraduates.

Learning strategies are an important component of self-regulated learning. Learners are expected to use multiple strategies appropriately. This study focused on metacognitive knowledge in the use of learning strategies and attempted to clarify the hierarchical nature of multiple knowledge. Furthermore, the study provided suggestions that could lead to further efficient acquisition of learning strategies. Responses were obtained from 184 Japanese university students regarding the degree of strategy use, knowledge regarding strategy, and perceived benefit and cost of 28 reading strategies. Results of the hierarchical Bayesian modeling showed that strategy use was influenced by knowledge regarding strategy and perceived benefit and cost. Furthermore, the effects of perceived benefit and cost were lower in the absence of knowledge regarding strategy. This implies that to use a learning strategy, the learner must first be aware of it and the degree to which it is used (apart from its theoretical usefulness) is determined by subjective benefit and cost. Therefore, in classroom situations, it is desirable to explicitly teach not only the course content but also strategies appropriate for learning the content. Dependence of the effects of perceived benefit and cost of strategy use on the presence or absence of knowledge regarding strategy suggests a hierarchy of metacognitive knowledge regarding usage of learning strategies.

Genetic and antigenic analyses of H5N8 and H5N1 subtypes high pathogenicity avian influenza viruses isolated from wild birds and poultry farms in Japan in the winter of 2021-2022.

In the winter of 2021-2022, multiple subtypes (H5N8 and H5N1) of high pathogenicity avian influenza viruses (HPAIVs) were confirmed to be circulating simultaneously in Japan. Here, we phylogenetically and antigenically analyzed HPAIVs that were isolated from infected wild birds, an epidemiological investigation of affected poultry farms, and our own active surveillance study. H5 subtype hemagglutinin (HA) genes of 32 representative HPAIV isolates were classified into clade 2.3.4.4b lineage and subsequently divided into three groups (G2a, G2b, and G2d). All H5N8 HPAIVs were isolated in early winter and had HA genes belonging to the G2a group. H5N1 HPAIVs belong to the G2b and G2d groups. Although G2b viruses were widespread throughout the season, G2d viruses endemically circulated in Northeast Japan after January 2022. Deep sequence analysis showed that the four HPAIVs isolated at the beginning of winter had both N8 and N1 subtypes of neuraminidase genes. Environmental water-derived G2a HPAIV, A/water/Tottori/NK1201-2/2021 (H5N8), has unique polymerase basic protein 1 and nucleoprotein genes, similar to those of low pathogenicity avian influenza viruses (LPAIVs). These results indicate that multiple H5 HPAIVs and LPAIVs disseminated to Japan via transboundary winter migration of wild birds, and HPAIVs with novel gene constellations could emerge in these populations. Cross-neutralization test revealed that G2a H5N8 HPAIVs were antigenically distinct from a G2b H5N1 HPAIV, suggesting that antibody pressure in wild birds was involved in the transition of the HPAIV groups during the season.

New high-throughput screening method for Chinese hamster ovary cell lines expressing low reduced monoclonal antibody levels: application of a system controlling the gas phase over cell lysates in miniature bioreactors and facilitating multiple sample setup.

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3 L, and 1000 L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP+ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells.

Effects of intramolecular chain conformation on the hydration and miscibility of polyethylene glycol in water studied by means of polymer reference interaction site model theory.

To examine the conventional idea that the gauche conformation of the OCCO dihedral angle promotes the dissolution of polyethylene glycol (PEG) in water through strong hydration, the thermodynamic properties of liquid mixtures of PEG and water were studied by means of polymer reference interaction site model (PRISM) theory. The intramolecular correlation functions required as input for PRISM theory were calculated by the generator matrix method, accompanied by changes in the distribution of dihedral angles. In the infinite dilution limit, the increased probability of gauche conformation of the OCCO dihedral angles stabilizes the hydration of PEG through enhanced hydrogen bonding between the ether oxygen of PEG and water. The mixing Gibbs energies of the liquid mixtures were also calculated in the whole concentration range based on the Gibbs-Duhem equation, as per our recent proposal. A liquid-liquid phase separation was observed when all the dihedral angles of PEG were in the trans conformation; for the liquid mixture to be miscible in the whole concentration range, the introduction of the OCCO gauche conformation was found to be indispensable. The above theoretical results support the conventional idea that the OCCO gauche conformation is important for the high miscibility of PEG and water.

Multiple infections with H5N8 subtype high pathogenicity avian influenza viruses in a feral mallard.

During the 2020-2021 winter, Eurasian countries experienced large outbreaks caused by the clade 2.3.4.4b H5N8 subtype high pathogenicity avian influenza viruses (HPAIVs) in the wild bird populations. At least seven gene constellations have been found in the causal HPAIVs. When and where the various HPAIVs emerged remains unclear. Here, we successfully cloned H5N8 HPAIVs with multiple gene constellations from a tracheal swab of a dead mallard found at its wintering site in Japan in January 2021. According to their phylogeny, the bird was most likely co-infected with the E2 and E3 genotype clade 2.3.4.4b HPAIVs. The result indicates that feral waterbirds can be infected with multiple HPAIVs, and shed an HPAIV with novel gene constellation in Southern wintering sites.

[A Case of Laparoscopic Abdominoperineal Resection with TpTME for Adenocarcinoma of Anal Canal with Pagetoid Spread].

A man in his 80s was referred to our hospital with the chief complaint of perianal erosion. Colonoscopy revealed a peripheral flat lesion in the anal canal. Since immunohistological examination showed positive for CK20 and negative for GCDFP15, we made a preoperative diagnosis of anal canal cancer with Pagetoid spread. It was diagnosed as cT1bN0M0, cStage Ⅰ by TNM classification, and laparoscopic abdominoperineal resection with TpTME was performed. Negative biopsy of the perianal skin was confirmed both preoperation and during the operation. The postoperative course was uneventful, and no urinary dysfunction was observed. The patient was discharged 15 days after the operation. The histopathological diagnosis was negative margin. The patient is alive without recurrence 1 year after the operation. Adenocarcinoma of anal canal with Pagetoid spread is rare, and differentiation from Paget's disease is important for determining treatment policy. By conducting a detailed examination of the extent of tumor progression and using TpTME together, it was possible to perform surgery that both secured the CRM and preserved urinary function.

Phase equilibrium of three-component liquid systems composed of water, alcohol, and sodium chloride studied by the reference interaction-site model integral equation theory.

A theoretical method for calculating the thermodynamic properties and phase equilibria of a binary liquid mixture using the reference interaction-site model (RISM) integral equation theory, which we had proposed recently, was extended to ternary liquid systems containing salt. A novel dielectric correction of the RISM theory for a mixture of solvents was also proposed. The theory was applied to mixtures composed of water, alcohol, and NaCl, where the alcohol was either methanol or ethanol. The decrease in NaCl solubility with increasing alcohol molar fractions in the solvent was calculated. In the ethanol system, the theory yielded salt-induced liquid-liquid phase separation, which was observed experimentally in a ternary mixture of water, 1-propanol, and NaCl. The phase diagram of the ternary system was determined theoretically.

Foot extension and retraction in the clam Calyptogena okutanii without any Keber's valve: an inflatable fastener bag model.

In order to investigate the foot manipulation of a clam without a Keber's valve, Calyptogena okutanii was examined by light microscopy, magnetic resonance imaging and computed tomography. The foot chamber was divided into two compartments by a dense muscle fastener zone (FZ) comprising a pedal artery and sinuses in the mid-sagittal plane in between muscles running in the anterior-posterior oblique direction. The distal part of the foot chamber (inflatable fastener bag, IFB) had a loose superficial muscle layer. The proximal part of the foot chamber (visceral reservoir, VR) was covered by a dense superficial muscle layer. The outlet of the VR was connected with the hinge ligament duct, consisting of the hinge ligament, a pair of shells and the pericardium. Based on these anatomical structures, foot extension starts from contraction of muscles in the FZ, so that flow in the FZ is stopped. Then, the superficial muscles of the foot contract, and the pressure of the IFB increases so that the foot can extend. Foot retraction starts from the relaxation of muscles in the FZ so that the hemolymph returns to the VR. The hinge ligament duct allows a constant return flow from the foot chamber to the gills and the heart. The heart rate and the flow in the FZ, which decreased and increased during the foot extension and retraction, respectively, supported this model. In conclusion, the FZ of Calyptogena okutanii could be an alternative to Keber's valve in Anodonta, playing a similar role.

Clinical effect of home enteral tube feeding supplementation on nutritional status after esophagectomy with retrosternal gastric tube reconstruction.

Body weight loss and poor nutritional status are frequently observed after esophageal cancer surgery. The aim of this study was to pilot an investigation on the impact of home enteral tube feeding supplementation (HES) for up to 3 months after esophageal cancer surgery. We retrospectively reviewed consecutive 67 esophageal cancer patients who underwent esophagectomy with gastric tube reconstruction. We started HES from April 2017. The patients were divided into 2 groups. Among 67 patients, 40 patients underwent HES between April 2017 and November 2020 (HES group). Other 27 patients who underwent esophagectomy between January 2012 and March 2017 were not administered HES (C group). Thereafter, multiple factors concerning patient nutritional status at long-term follow-up were evaluated. The baseline characteristics were balanced between the two groups. There were no significant differences in nutritional status scores before esophagectomy. The percentage weight loss was less in the HES group compared with the C group both at 3 months and 1 year after surgery: 7.3% (-7.6 to 15.2), 7.7% (-4 to 13.9) in the HES group and 10.6% (-3.6 to 29.1), 10.8% (-5.8 to 20.0) in C group (P < 0.05, P < 0.05). In the patients with anastomotic stenosis, the percentage weight loss was less in the HES group compared with the C group: 7.2% (2.0-14.9) and 14.6% (6.2-29.1), P < 0.05. HES may improve early weight loss in postesophagectomy patients.

Study of phase equilibria and thermodynamic properties of liquid mixtures using the integral equation theory: Application to water and alcohol mixtures.

A theoretical method for calculating the thermodynamic properties and phase equilibria of liquid-liquid mixtures using the integral equation theory is proposed. The solvation chemical potentials of the two components are evaluated by the integral equation theory and the isothermal-isobaric variation of the total density with composition is determined to satisfy the Gibbs-Duhem relation. Given the density of a pure component, the method can calculate the densities of the mixture at any composition. Furthermore, it can treat the phase equilibrium without thermodynamic inconsistency with respect to the Gibbs-Duhem relation. This method was combined with the reference interaction-site model integral equation theory and applied to mixtures of water + 1-alcohol by changing the alcohol from methanol to 1-butanol. The destabilization of the mixing Gibbs energy by increasing the hydrophobicity of the alcohol and demixing of the water-butanol mixture were reproduced. However, quantitative agreement with experiments is not satisfactory, and further improvements of the integral equation theory and the molecular models are required.

Effects of molecular shape and flexibility on fast sound of organic liquids.

Inelastic x-ray scattering spectra of four organic liquids, n-hexane, cyclohexane, ethylene glycol dimethyl ether, and 1,4-dioxane, were measured, and the sound velocity in the nm-1 wavenumber and meV energy regimes was determined. Compared with the corresponding values in the hydrodynamic limit, the sound velocity in the nm-1 regime was faster, and the positive dispersion of the longitudinal modulus was stronger in liquids composed of ring structures (cyclohexane and 1,4-dioxane) than in those of linear chain structures (n-hexane and ethylene glycol dimethyl ether). Molecular dynamics simulation of n-hexane and cyclohexane was also performed. The difference in the positive dispersion of the longitudinal modulus was reproduced by simulation, and it was elucidated by the difference in the longitudinal modulus in the q = 0 limit and the THz frequency regime. The excess part of the longitudinal modulus from the hydrodynamic limit was further divided into various contributions, and the smaller excess modulus of n-hexane was mainly ascribed to two reasons. The first one is that the shear modulus of n-hexane is smaller in the THz regime, and the second one is that the positive dispersion of the bulk modulus due to the vibrational energy relaxation is weaker. The second mechanism was further interpreted in terms of the fast vibrational energy relaxation of intramolecular modes associated with the chain deformation of n-hexane.

A paradigm shift in learning strategy research: Illustration and example of a within-person examination.

Although research on learning strategies has contributed to improving learners' performance, most studies only described the characteristics of learners. These approaches have focused on inter-individual differences and their correlations, making it difficult to examine the process of selecting learning strategies in individuals. To examine the correlations within individual learners, such as the variables that influence the use of a strategy by a specific learner, two surveys were conducted in this study. Perceived cost and perceived benefit of using each strategy as the influential variables on choosing a strategy were adopted with reference to decision-making research. Perceived cost consisted of the time perspective for learning outcomes by the learning strategy and the method of learning strategy implementation. In Study 1, cognitive strategies that have a direct impact on the acquisition of learning content were measured, and metacognitive strategies that monitor and regulate one's own learning behavior were examined in Study 2. The variables were treated for each individual to calculate intra-class and within-person correlations, although data were collected using a typical questionnaire survey in learning strategy studies. Hierarchical Bayesian modeling was conducted with use of the learning strategy as the objective variable and the perceived costs and benefits as explanatory variables. The analysis revealed that, in terms of cognitive strategies (Study 1), learners avoided using learning strategies perceived to have a high cost in the short term; however, they used cognitive strategies regardless of the circumstances. Furthermore, regarding metacognitive strategies (Study 2), students avoided using learning strategies that they perceived as costly to use in the short term and used them only when they suited the circumstances. On the other hand, in metacognitive strategies (Study 2), students avoided using learning strategies that they perceived as costly to use for the next test and use as appropriate for the situation. Focusing on within-individual variance and correlation made the interpretation of factors that influence the choice of learning strategies more intuitive and provided more suggestions for educational practice.

A novel, simple, and dedicated device for endoscopic mucosal defect closure.

Objectives: Endoscopic submucosal dissection (ESD) has become popular, but complications such as postoperative bleeding remain an issue. Although some methods of closing a mucosal defect with a snare and clips have been reported to be effective and safe, the snare is not a dedicated device, and the procedure is difficult and time-consuming. We aimed to find an alternative method for defect closure after ESD by developing a dedicated device. Methods: We have improved five prototypes. The load on the stopper when starting to tighten and loosen a loop and the maximum load on the stopper and the movement distance of the thread when sliding the stopper were measured five times for each prototype. With the 5th prototype, we finalized the design and named it FLEXLOOP. Additionally, the material and shape of the outer tube were improved. Then, the usability of FLEXLOOP was evaluated in pigs. The operation time for closing mucosal defects with the snare or FLEXLOOP was measured five times. Results: We made FLEXLOOP, which had a lower load when sliding and a higher load when loosening than the snare. The improvement of the outer tube significantly reduced the load on the sheath when sliding it. We confirmed the feasibility of mucosal defect closure with FLEXLOOP in pigs. The median operation time was 563 s (range 340-679 s) with the snare and 355 s (range 303-455 s) with FLEXLOOP (p = 0.047). Conclusions: FLEXLOOP can be a promising option for defect closure after ESD.

Surgical invasiveness and lymphadenectomy in robotic and laparoscopic gastrectomy: A retrospective study with propensity-score matching.

BACKGROUND: There is no consensus amongst comparative studies about the advantages of robotic over laparoscopic surgeries for gastric cancer (GC). We compared invasiveness and lymph node dissection between robotic and laparoscopic gastrectomies (RG and LG). METHODS: We retrospectively reviewed the medical records of 215 consecutive patients with GC who underwent RG or LG with lymphadenectomy from January 2011-December 2020. Propensity score matching analysis was performed to control selection bias. RESULTS: The RG group had less operative blood loss (P = 0.0005) and higher C-reactive protein levels on postoperative day 1 (P = 0.0006) than the LG group. When analyzing the specific sites of dissected lymph nodes, station groups of supra-pancreatic and lesser curvature areas accounted for this difference (P = 0.0073 and 0.0362, respectively). CONCLUSIONS: RG demonstrated lesser intraoperative bleeding, less of a postoperative inflammatory response, and a higher proportion of lymph node removal than LG, suggesting that it is a better surgical and oncological procedure.

Comparison between longitudinal viscoelastic relaxation and sound dispersion of molecular liquids on the molecular scale.

Molecular dynamics simulation on some molecular liquids was performed to study sound dispersion on the molecular scale. The sound velocity was determined from the intermediate scattering function, and the relation between the longitudinal modulus and frequency was compared with the frequency-dependent longitudinal modulus in the q = 0 limit evaluated by the Kubo-Green theory. The sound dispersion of a monoatomic liquid up to qσ ≅ 2 was almost quantitatively explained by the viscoelasticity in the q = 0 limit when the wavenumber dependence of the heat capacity ratio was taken into account. The situation was similar for a polyatomic molecular liquid for which the intramolecular degrees of freedom were fixed. For a polyatomic liquid with intramolecular degrees of freedom, the sound dispersion on the molecular scale was connected to the high-frequency limit of the ultrasonic relaxation mode assigned to the vibrational energy relaxation. After subtracting the contribution of the vibrational energy relaxation, both the longitudinal viscoelasticity and the sound dispersion depended little on the presence of intramolecular degrees of freedom.

Reference Values for Weight Loss During 1 Year After Sleeve Gastrectomy: a Multicenter Retrospective Study in Japan.

INTRODUCTION: This study aimed to provide reference values for the percentage total weight loss during 1 year after laparoscopic sleeve gastrectomy associated with primary response in Japan. METHODS: This multicenter retrospective study comprised 248 patients with severe obesity who underwent laparoscopic sleeve gastrectomy at five institutions in Japan. A percentage total weight loss < 20% at 1 year was defined as primary non-response. Parsimonious predictive models were developed based on the results of multiple regression analyses. A receiver operating characteristic curve analysis was used to assess the discriminative performance for primary non-response. RESULTS: The median age, initial body mass index, and percentage total weight loss at 1 year were 41 years, 41.4 kg/m2, and 30.1%, respectively. There were 28 (11.3%) primary non-responders. For discriminating primary non-responders, the areas under the receiver operating characteristic curve of the parsimonious model and actual percentage of total weight loss at 3 months after laparoscopic sleeve gastrectomy were 0.846 and 0.803, respectively. Cutoffs for the predicted percentage total weight loss using the model and actual value of percentage total weight loss at 3 months attaining 80% sensitivity were 30% and 22%, and those attaining 98% specificity were 22% and 15%, respectively. CONCLUSIONS: Reference values for the percentage total weight loss at 3 months were obtained using the predictive model and actual value of percentage total weight loss at 3 months. Patients at risk for primary non-response can be determined at 3 months with these values, which can help in considering earlier interventions.

Volume viscosity and ultrasonic relaxation of ethanol-water mixtures studied by molecular dynamics simulations.

The volume viscosity of ethanol-water mixtures at various compositions was calculated by means of equilibrium molecular dynamics simulations, and the results were compared with acoustic experiments. The volume viscosity exhibited a strong maximum around the ethanol mole fraction of 0.27, which is in agreement with experiments. The relaxation in the 100 ps regime, which had been revealed by ultrasonic spectroscopy, was also reproduced. Analysis of the two-body density and the adiabatic pressure fluctuation demonstrated that the large volume viscosity of the mixture originates from the long-range concentration fluctuation.