Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible regions located outside the CDR (complementarity determining region) and the interface between the heavy-chain and light-chain variable regions. We then designed a thermostable mutant and evaluated it by means of a short MD simulation (3 runs of 50 ns) based on reductions in the root-mean-square fluctuation (RMSF) values and formation of new hydrophilic interactions around the weak spot. Finally, we designed the VL-R66G mutant by applying our strategy to scFv derived from trastuzumab. Trastuzumab scFv variants were prepared by using an Escherichia coli expression system, and the melting temperature-measured as a thermostability index-was 5 °C higher than that of the wild-type trastuzumab scFv, while the antigen-binding affinity was unchanged. Our strategy required few computational resources, and would be applicable to antibody drug discovery.

In silico-labeled ghost cytometry.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

Relationship between coffee and green tea consumption and all-cause mortality in a cohort of a rural Japanese population.

We conducted a cohort study to investigate the effects of coffee and green tea consumption on all-cause mortality in a rural Japanese population. Data were obtained from 2,855 men and women aged 40-79 years in 1989, and during the subsequent 9.9 years of follow-up. Using the Cox regression model to adjust for potential confounding factors, we calculated the multivariate hazard ratios of death from all causes separately for men and women. The multivariate hazard ratio of mortality for men who consumed two or more cups of coffee per day, compared with those who consumed less than half a cup per day, was 0.43 (95% confidence interval, 0.30-0.63), and the ratio for those who consumed half to one cup of coffee per day was 0.70 (95% confidence interval, 0.52-0.94). Exclusion of subjects with less than 5 years of follow-up did not substantially change the findings. No other statistically significant associations were identified between consumption of the two beverages and all-cause mortality. For men, multivariate hazard ratios of death from apoplexy showed a significant inverse association with increasing coffee consumption. The effects of habitual coffee consumption and its related factors on health in Japan need to be studied in greater detail.