KRAS is a small GTPase essential for cell signaling, and the G12C mutation acts as a key oncogenic driver in multiple cancers. First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, have shown clinical efficacy, but are limited by acquired resistance due to secondary mutations. In this study, we investigated the impact of secondary mutations (Y96D, Y96S, G13D, and Q99L) on the binding efficacy of sotorasib, adagrasib, and the next-generation inhibitor (MK-1084) using molecular dynamics simulations, binding free energy calculations, and dynamic protein-ligand interaction analysis. Our study revealed that each secondary mutant variant exhibited variations in the degree of resistance to the inhibitors. Two major resistance patterns were identified: direct and indirect. Our analyses revealed that Y96 mutations directly disrupt inhibitor binding, conferring high resistance to all three inhibitors, whereas G13D and Q99L indirectly alter the binding environment by influencing other residues, resulting in variable resistance profiles. This study provides detailed molecular insights into resistance mechanisms to support the rational design of more robust KRAS G12C-targeted therapies.

KRAS G12C inhibitors such as sotorasib and adagrasib are effective targeted therapies but often fail due to resistance from secondary mutations. Using molecular dynamics simulations, binding free energy calculations, and protein–ligand interaction analyses, we investigated how Y96D, Y96S, G13D, and Q99L mutations affect the binding of sotorasib, adagrasib, and the next-generation inhibitor MK-1084. Our results reveal mutation-specific resistance mechanisms. Y96 mutations directly disrupt inhibitor binding, leading to strong resistance, while G13D and Q99L indirectly reshape the binding environment, producing variable resistance profiles. These insights support the rational design of more robust KRAS G12C inhibitors.

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The aim of this study was to evaluate StepAn as an automated approach for quantitative gait analysis in rodents. We sought to determine whether this video-based approach, which uses standard recordings from devices like smartphones, could sensitively detect motor deficits in a pharmacological model of Parkinsonian bradykinesia. Wistar rats received a single intraperitoneal injection of saline (control), 0.15 mg/kg haloperidol, or 0.30 mg/kg haloperidol. One hour post-injection, gait was analyzed using both the traditional manual “Footprints” test and the StepAn-based automated video analysis, which performs paw detection and stride length calculation from video recordings. Both analysis methods detected a significant reduction in average stride length in haloperidol-treated rats compared to controls (p<0.001), confirming the expected bradykinetic phenotype. No significant difference was found between the two haloperidol doses. Automated analysis provided equivalent mean values but demonstrated superior precision, evidenced by significantly lower measurement variability compared to manual scoring. We evaluated StepAn as a precise and hardware-flexible tool for quantitative gait assessment. Its ability to detect drug-induced Parkinsonian gait deficits comparable to established methods supports its use for objective locomotor analysis in preclinical research.

Rats received saline or haloperidol (0.15 or 0.30 mg/kg, i.p.), and gait was assessed 1 h later using either the conventional Footprints method or automated video analysis with StepAn. Both approaches detected haloperidol-induced stride shortening, confirming a bradykinetic gait phenotype. Automated analysis produced results comparable to manual scoring while showing lower measurement variability and compatibility with standard recording devices, including smartphones and webcams. These findings support StepAn as an objective and accessible tool for quantitative gait assessment in preclinical studies.

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Bioluminescence is an oxidation-mediated chemical reaction involving the luciferase enzyme and the luciferin substrate. To date, various artificial luciferases have been developed, and their luminescence properties have been investigated. However, very few studies have focused on the solvent environment in which these luminescent reactions occur, and the details of their influence on luminescent phenomena remain unclear. In this study, we investigated the effects of various surfactants on the activity of the luciferase picALuc. To this end, we analyzed the effects of adding diverse surfactants on the luminescence properties of the luciferase picALuc. The results showed that the addition of a surfactant enhanced the luminescence intensity and extended the duration of luminescence. Dynamic light scattering measurements and microscopic observations revealed that micelle-like particles were formed by the surfactant, which suppressed luminescence inhibition. This study provides our original method for adding surfactants to enzymatic reactions, such as those involving hydrophobic substrates.

Surfactants affect the luciferase reaction, increasing luminescence intensity and duration by forming micellar particles with the inhibitory coelenterazine metabolites and alleviating their inhibition. Thus, adding surfactants provides a useful strategy for controlling enzyme reactions.

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Hyperthermal sarcomeric oscillations (HSOs) expose rapid sarcomere-level motion in living cardiomyocytes and provide a mesoscopic window between actomyosin activity and robust cellular contraction. I reanalyzed high-speed sarcomere-length recordings from five consecutive sarcomeres in each of seven neonatal rat cardiomyocytes. During HSOs, local phase relations became trackable through most of the oscillatory segment (valid fraction, 0.298 before warming and 0.956 during HSOs; paired Wilcoxon P=0.0156). Neighboring-sarcomere reconfiguration was dominated by one-link switches, in which one adjacent-pair relation changed while the other three were maintained (216/230 HSO phase transitions), and anti-phase-rich occupancy increased from 0.254 to 0.509 (P=0.0156). I then measured event-local relative internal length redistribution. For each reach-qualified one-link event, compensation reach, S, was defined as the expected sarcomere-index distance between relative shortening and relative lengthening. The same directed IAAI-to-IAII switch was accompanied by short-reach redistribution in one event (S=1.29) and cross-chain redistribution in another (S=2.88). Across 248 reach-qualified events, S increased with the pre-event number of I-type links, with a cell-fixed slope of 0.148 span units per added I-link supported by cell-blocked permutation and cell-cluster bootstrap analyses. Thus, HSO reveals a mesoscale organizing process in which a local switch in neighboring-sarcomere synchrony is linked to spatially distributed relative internal length redistribution whose reach is shaped by the pre-event phase context.

Hyperthermal sarcomeric oscillations reveal a mesoscopic organizing process in living neonatal cardiomyocytes. Neighboring-sarcomere phase relations are updated mainly by one-link switches, and each measurable switch recruits internal length compensation with a spatial reach shaped by the pre-event phase context.

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Bioluminescence imaging (BLI) detects light emission from samples expressing luciferase in the presence of luciferin. Unlike fluorescence imaging, BLI does not require excitation light, thereby avoiding phototoxicity in living cells and photobleaching of labels. However, the intrinsically slow turnover of the luciferin-luciferase reaction often results in low luminescence intensity, which degrades image quality and limits practical applications, particularly in high-magnification optical microscopy of cells. In this study, we demonstrate that image denoising provides an effective strategy to overcome this fundamental limitation of BLI. We developed a physics-based noise model that incorporates fixed-pattern noise, blooming noise, readout noise, quantization noise, and Poisson shot noise, enabling accurate estimation of noise parameters encountered in EMCCD-based microscopy. In the denoising pipeline, raw noisy images were first corrected for the fixed-pattern and blooming noises, followed by restoration using Uformer neural network trained with paired ground-truth images and synthetically generated noisy images. Quantitative evaluation using the peak signal-to-noise ratio (PSNR) and structural similarity index measure (SSIM) showed that denoised images derived from raw data acquired with exposure times of ≤100 ms achieved PSNR and SSIM values comparable to those of raw images acquired with exposure times of 1–3 s. These results indicate that the proposed denoising approach substantially extends the practical limit of high-magnification BLI.

Bioluminescence images of organelles at high magnification such as mitochondria labeled with luciferase are so dim that they can be clearly visualized only at considerably extended exposure time of, e.g., longer than 1 s. The present paper reports an image denoising technique involving the correction of blooming and fixed pattern noise (FPN) followed by noise reduction using a convolutional neural network (CNN) of Uformer. The present technique is able to restore a clean image with as high quality as that of a corresponding raw image acquired at a 30-fold longer exposure time.

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