Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.

Autonomy support encourages use of more-affected arm post-stroke.

BACKGROUND: Autonomy support, which involves providing individuals the ability to control their own behavior, is associated with improved motor control and learning in various populations in clinical and non-clinical settings. This study aimed to investigate whether autonomy support combined with an information technology (IT) device facilitated success in using the more-affected arm during training in individuals with stroke. Consequently, we examined whether increased success influenced the use of the more-affected arm in mild to moderate subacute to chronic stroke survivors. METHODS: Twenty-six participants with stroke were assigned to the autonomy support or control groups. Over a 5-week period, training and test sessions were conducted using the Individualized Motivation Enhancement System (IMES), a device developed specifically for this study. In the autonomy support group, participants were able to adjust the task difficulty parameter, which controlled the time limit for reaching targets. The control group did not receive this option. The evaluation of the more-affected arm's use, performance, and impairment was conducted through clinical tests and the IMES. These data were then analyzed using mixed-effect models. RESULTS: In the IMES test, both groups showed a significant improvement in performance (p < 0.0001) after the training period, without any significant intergroup differences (p > 0.05). However only the autonomy support group demonstrated a significant increase in the use of the more-affected arm following the training (p < 0.001). Additionally, during the training period, the autonomy support group showed a significant increase in successful experiences with using the more-affected arm (p < 0.0001), while the control group did not exhibit the same level of improvement (p > 0.05). Also, in the autonomy support group, the increase in the use of the more-affected arm was associated with the increase in the successful experience significantly (p = 0.007). CONCLUSIONS: Combining autonomy support with an IT device is a practical approach for enhancing performance and promoting the use of the more-affected upper extremity post-stroke. Autonomy support facilitates the successful use of the more-affected arm, thereby increasing awareness of the training goal of maximizing its use. TRIAL REGISTRATION: The study was registered retrospectively with the Clinical Research Information Service (KCT0008117; January 13, 2023; https://cris.nih.go.kr/cris/search/detailSearch.do/23875 ).

Metabolite Profiling to Evaluate Metabolic Changes in Genetically Modified Protopanaxadiol-Enriched Rice.

Event DS rice producing protopanaxadiol (PPD) has been previously developed by inserting Panax ginseng dammarenediol-II synthase gene (PgDDS) and PPD synthase gene (CYP716A47). We performed a gas chromatography-mass spectrometry (GC-MS)-based metabolomics of the DS rice to identify metabolic alterations as the effects of genetic engineering by measuring the contents of 65 metabolites in seeds and 63 metabolites in leaves. Multivariate analysis and one-way analysis of variance between DS and non-genetically modified (GM) rice showed that DS rice accumulated fewer tocotrienols, tocopherols, and phytosterols than non-GM rice. These results may be due to competition for the same precursors because PPDs in DS rice are synthesized from the same precursors as those of phytosterols. In addition, multivariate analysis of metabolic data from rice leaves revealed that composition differed by growth stage rather than genetic modifications. Our results demonstrate the potential of metabolomics for identifying metabolic alterations in response to genetic modifications.

Pathogenic Role of RAGE in Tau Transmission and Memory Deficits.

BACKGROUND: In tauopathies, brain regions with tau accumulation strongly correlate with clinical symptoms, and spreading of misfolded tau along neural network leads to disease progression. However, the underlying mechanisms by which tau proteins enter neurons during pathological propagation remain unclear. METHODS: To identify membrane receptors responsible for neuronal propagation of tau oligomers, we established a cell-based tau uptake assay and screened complementary DNA expression library. Tau uptake and propagation were analyzed in vitro and in vivo using a microfluidic device and stereotactic injection. The cognitive function of mice was assessed using behavioral tests. RESULTS: From a genome-wide cell-based functional screening, RAGE (receptor for advanced glycation end products) was isolated to stimulate the cellular uptake of tau oligomers. Rage deficiency reduced neuronal uptake of pathological tau prepared from rTg4510 mouse brains or cerebrospinal fluid from patients with Alzheimer's disease and slowed tau propagation between neurons cultured in a 3-chamber microfluidic device. RAGE levels were increased in the brains of rTg4510 mice and tau oligomer-treated neurons. Rage knockout decreased tau transmission in the brains of nontransgenic mice after injection with Alzheimer's disease patient-derived tau and ameliorated memory loss after injection with GFP-P301L-tau-AAV. Treatment of RAGE antagonist FPS-ZM1 blocked transsynaptic tau propagation and inflammatory responses and alleviated cognitive impairment in rTg4510 mice. CONCLUSIONS: These results suggest that in neurons and microglia, RAGE binds to pathological tau and facilitates neuronal tau pathology progression and behavioral deficits in tauopathies.

Effect of changing the lying posture angle by changing the bed posture on the average pressure, maximum pressure, and pressure area in the hip region.

BACKGROUND: An electric bed can easily change posture from a lying position and was effective in preventing pressure ulcer. OBJECTIVE: This study aimed to identify the optimal posture for the prevention of pressure ulcers by analyzing pressure changes applied to the pelvic region. METHODS: Pressure changes resulting from lateral rotations of the body using an electronic adjustable bed and changes in the posture and angles of the trunk and knees were assessed. Twelve conditions with varying angles of the trunk and knees (15-35∘ in 5∘ increments) and varying lateral angles (20-35∘ in 5∘ increments) were tested. The pressure (maximum and average) and contact area in the pelvic region of 20 individuals without disabilities were calculated. RESULTS: The conditions in which the average and maximum pressures did not increase according to the increase in angle were 25∘ for the upper body and knee angles and 35∘ for the side. CONCLUSIONS: The body pressure changed according to the posture rather than according to physical characteristics. Lateral rotation combined with changes in the angles of the trunk and knees effectively prevented pressure ulcers. Changes in the posture at various angles prevented an increased pressure on the body.

Investigating Activity Recognition for Hemiparetic Stroke Patients Using Wearable Sensors: A Deep Learning Approach with Data Augmentation.

Measuring the daily use of an affected limb after hospital discharge is crucial for hemiparetic stroke rehabilitation. Classifying movements using non-intrusive wearable sensors provides context for arm use and is essential for the development of a home rehabilitation system. However, the movement classification of stroke patients poses unique challenges, including variability and sparsity. To address these challenges, we collected movement data from 15 hemiparetic stroke patients (Stroke group) and 29 non-disabled individuals (ND group). The participants performed two different tasks, the range of motion (14 movements) task and the activities of daily living (56 movements) task, wearing five inertial measurement units in a home setting. We trained a 1D convolutional neural network and evaluated its performance for different training groups: ND-only, Stroke-only, and ND and Stroke jointly. We further compared the model performance with data augmentation from axis rotation and investigated how the performance varied based on the asymmetry of movements. The joint training of ND + Stroke yielded an increased F1-score by a margin of 31.6% and 10.6% compared to ND-only training and Stroke-only training, respectively. Data augmentation further enhanced F1-scores across all conditions by an average of 11.3%. Finally, asymmetric movements decreased the F1-score by 25.9% compared to symmetric movements in the Stroke group, indicating the importance of asymmetry in movement classification.

Active Aging In Long-Term Care Facilities In Korea : Beyond The Lexical Meaning.

The discourse of active aging, as introduced by the WHO, aims at optimizing older adults' opportunities for health, participation, and security that could eventually enhance their social integration and quality of life. Considering that even those with frailty could strive for active aging in the given circumstances, we examined the meaning of active aging in long-term care settings and care strategies to promote it based on the WHO's framework. We conducted interviews with a total of 35 participants. The interpretative analyses revealed that the activities taken place in LTCFs have various scopes depending on older adults' physical and cognitive functional ability, and it captures the forms of activities that go beyond its lexical meaning. By defining being "active," the present findings could contribute to an understanding of how the three elements of active aging can be carried out in LTCFs.

Cisplatin fastens chromatin irreversibly even at a high chloride concentration.

Cisplatin is one of the most potent anti-cancer drugs developed so far. Recent studies highlighted several intriguing roles of histones in cisplatin's anti-cancer effect. Thus, the effect of nucleosome formation should be considered to give a better account of the anti-cancer effect of cisplatin. Here we investigated this important issue via single-molecule measurements. Surprisingly, the reduced activity of cisplatin under [NaCl] = 180 mM, corresponding to the total concentration of cellular ionic species, is still sufficient to impair the integrity of a nucleosome by retaining its condensed structure firmly, even against severe mechanical and chemical disturbances. Our finding suggests that such cisplatin-induced fastening of chromatin can inhibit nucleosome remodelling required for normal biological functions. The in vitro chromatin transcription assay indeed revealed that the transcription activity was effectively suppressed in the presence of cisplatin. Our direct physical measurements on cisplatin-nucleosome adducts suggest that the formation of such adducts be the key to the anti-cancer effect by cisplatin.

Antigen-Presenting, Self-Assembled Protein Nanobarrels as an Adjuvant-Free Vaccine Platform against Influenza Virus.

Although naturally occurring, self-assembled protein nanoarchitectures have been utilized as antigen-delivery carriers, and the inability of such carriers to elicit immunogenicity requires additional use of strong adjuvants. Here, we report an immunogenic Brucella outer membrane protein BP26-derived nanoarchitecture displaying the influenza extracellular domain of matrix protein-2 (M2e) as a vaccine platform against influenza virus. Genetic engineering of a monomeric BP26 containing four or eight tandem repeats of M2e resulted in a hollow barrel-shaped nanoarchitecture (BP26-M2e nanobarrel). Immunization with BP26-M2e nanobarrels induced a strong M2e-specific humoral immune response in vivo that was much greater than that of a physical mixture of soluble M2e and BP26, with or without the use of an alum adjuvant. An anti-M2e antibody generated by BP26-M2e nanobarrel-immunized mice specifically bound to influenza virus-infected cells. Furthermore, in viral challenge tests, BP26-M2e nanobarrels effectively protected mice from influenza virus infection-associated death, even without the use of a conventional adjuvant. A mechanism study revealed that both M2e-specific antibody-dependent cellular cytotoxicity and T cell responses are involved in the vaccine efficacy of BP26-M2e nanobarrels. These findings suggest that the BP26-based nanobarrel developed here represents a versatile vaccine platform that can be used against various viral infections.

Simultaneous determination of eight arginine-related metabolites in cellular extracts using liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of arginine and its pathway-related metabolites (ornithine, proline, citrulline, glutamate, agmatine, spermidine, and spermine) in cellular extracts. Cells were lysed and cellular proteins precipitated by the addition of acetonitrile followed by ultra-sonication. Supernatants were analyzed using a Chromolith High Resolution RP-18 endcapped column (100 × 4.6 mm, 1.15 µm, 150 Å), with mobile phases of 0.1% formic acid solution and 0.1% formic acid in acetonitrile. Detection was carried out in multiple reaction monitoring (MRM) mode. Calibration curves showed linearity (r2 > 0.99) for all metabolites over the calibration ranges used. The intra- and inter-day precision was less than 13.5%, and the accuracy was between 91.3 and 114.7%. The method developed in this study was successfully applied to measure arginine and its pathway-related metabolites, which are related to nitric oxide synthase/arginase pathways in mouse bone marrow-derived dendritic cells (BMDCs). The ability to simultaneously measure arginine and its pathway-related metabolites is valuable for better understanding local and systemic inflammatory processes.

Determination of motolimod concentration in rat plasma by liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

Motolimod (VTX-2337) is an agonist of toll-like receptor 8. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantifying motolimod in rat plasma and subsequently used in a pharmacokinetic study. Proteins were precipitated from plasma samples using acetonitrile prior to the analysis. GS-9620 was used as an internal standard. High-performance liquid chromatography was performed using a Spursil C18-EP column (3 µm, 50 × 2.1 mm). Aqueous ammonium formate and acetonitrile were used as the mobile phase. Motolimod was detected using an electrospray ionization interface under multiple reaction monitoring conditions in the positive ion mode. The developed method produced a linear correlation over a concentration range of 1-1000 ng/mL (r = 0.9944). Intra- and inter-day precision values ranged from 4.8%-10.7% (the lower limit of quantification precision value was 16.3 %), whereas intra- and inter-day accuracy values ranged from 0.3%-9.1 %. The mean recovery of motolimod from rat plasma was 109.4 %. Additionally, motolimod was found to be stable under various conditions (three freeze-thaw cycles, 6-h storage at room temperature, short- and long-term stability tests, and processing). The developed method was successfully used in a pharmacokinetic study in rats. Motolimod showed non-linear pharmacokinetics following its intravenous administration to rats at 0.6-6 mg/kg. Additionally, very low exposure (<1 %) was obtained following oral administration of the drug to rats. The results also showed that motolimod has a low metabolic stability in the liver microsomes and exhibits extensive binding to the plasma proteins.

Plasma metabolites as possible biomarkers for diagnosis of breast cancer.

Metabolomic approaches have been used to identify new diagnostic biomarkers for various types of cancers, including breast cancer. In this study, we aimed to identify potential biomarkers of breast cancer using plasma metabolic profiling. Furthermore, we analyzed whether these biomarkers had relationships with clinicopathological characteristics of breast cancer. Our study used two liquid chromatography-mass spectrometry sets: a discovery set (40 breast cancer patients and 30 healthy controls) and a validation set (30 breast cancer patients and 16 healthy controls). All breast cancer patients were randomly selected from among stage I-III patients who underwent surgery between 2011 and 2016. First, metabolites distinguishing cancer patients from healthy controls were identified in the discovery set. Then, consistent and reproducible metabolites were evaluated in terms of their utility as possible biomarkers of breast cancer. Receiver operating characteristic (ROC) analysis was applied to the discovery set, and ROC cut-off values for the identified metabolites derived therein were applied to the validation set to determine their diagnostic performance. Ultimately, four candidate biomarkers (L-octanoylcarnitine, 5-oxoproline, hypoxanthine, and docosahexaenoic acid) were identified. L-octanoylcarnitine showed the best diagnostic performance, with a 100.0% positive predictive value. Also, L-octanoylcarnitine levels differed according to tumor size and hormone receptor expression. The plasma metabolites identified in this study show potential as biomarkers allowing early diagnosis of breast cancer. However, the diagnostic performance of the metabolites needs to be confirmed in further studies with larger sample sizes.

Inhibition of Cytochrome P450 Activities by Sophora flavescens Extract and Its Prenylated Flavonoids in Human Liver Microsomes.

Sophora flavescens possesses several pharmacological properties and has been widely used for the treatment of diarrhea, inflammation, abscess, dysentery, and fever in East Asian countries. S. flavescens is a major source of prenylated flavonoids, such as sophoraflavone and kushenol. In this study, we examined the effects of S. flavescens extract and its prenylated flavonoids on cytochrome P450 (CYP) isoform activity in human liver microsomes. The extract inhibited CYP2C8, CYP2C9, CYP2C19, and CYP3A activities, with IC50 values of 1.42, 13.6, 19.1, and 50 µg/mL, respectively. CYP2B6 was only inhibited in human liver microsomes preincubated with the extract. CYP3A4 was more strongly inhibited by the extract in the presence of NADPH, suggesting that the extract may inhibit CYP2B6 and CYP3A4 via mechanism-based inactivation. Prenylated flavonoids also inhibited CYP isoforms with different selectivity and modes of action. Kushenol I, leachianone A, and sophoraflavone G inhibited CYP2B6, whereas kushenol C, kushenol I, kushenol M, leachianone A, and sophoraflavone G inhibited CYP3A4 via mechanism-based inhibition. Our results suggest that S. flavescens may contribute to herb-drug interactions when coadministered with drugs metabolized by CYP2B6, CYP2C8, CYP2C9, and CYP3A4.

Liquid chromatography-tandem mass spectrometry of recombinant human extracellular superoxide dismutase (rhSOD3) in mouse plasma and its application to pharmacokinetic study.

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.

Anticancer effects of a novel chroman analog in HeLa cells are associated with G2­phase arrest and mitochondrial­mediated apoptosis.

In the present study, the anticancer activity of 1­[(3S,4R)­2,2­dimethyl­3­oxo­4­(2­piperidonyl)chroman­6­yl]­3­phenylurea (S32) was investigated by testing its effect in vitro on the growth of HeLa cells. First, we showed that the IC50 value of S32 was ~70 µM by using WST­8 assay, and that it significantly inhibited the proliferation and viability of HeLa cells in a dose­dependent manner after 48 h. Morphological changes in apoptotic cells included cellular shrinkage and nuclear condensation. The results of [3H]­thymidine incorporation and flow cytometric analysis indicated that S32 induced inhibition of DNA replication and G2­phase cell cycle arrest. Moreover, S32 induced the levels of reactive oxygen species (ROS) and decreased the mitochondrial membrane potential (MMP) in a time­dependent manner. Using Annexin V­FITC/propidium iodide (PI) dual staining assay, we found that S32 noticeably increased early apoptosis in HeLa cells in a time­dependent manner. The result of western blot analysis showed that the apoptotic induction was associated with an increase in Bax levels and a decrease in Bcl­2 levels, which led to activation of caspase­8, ­9 and ­3. Taken together, our findings demonstrated that S32 induces mitochondrial­mediated apoptosis in HeLa cells and suggest that S32 has potential as an anticancer drug.

Antioxidative, anti-inflammatory, and matrix metalloproteinase inhibitory activities of 20(S)-ginsenoside Rg3 in cultured mammalian cell lines.

Ginsenoside Rg3 is one of ginsenosides that are the well-known bioactive principles of Panax ginseng. Among the two stereoisomeric forms of Rg3, 20(S)-ginsenoside Rg3 [20(S)-Rg3] is predominant. 20(S)-Rg3 is capable of suppressing the nitric oxide (NO), reactive oxygen species (ROS) and prostaglandin E2 (PGE2) productions induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells in a concentration-dependent manner. In the same stimulated macrophages, 20(S)-Rg3 was able to suppress matrix metalloproteinase-9 (MMP-9) activity and suppress cyclooxygenase-2 (COX-2) expression. It suppressed the production of some proinflammatory cytokines, such as TNF-α, IL-1ß and IL-6, and the cell mobility enhanced by LPS in the macrophage cells. 20(S)-Rg3 displayed suppressive effect on the ROS level but not on the NO level, and down-regulating effect on MMP-9 but not on MMP-2 in non-stimulated HaCat keratinocytes. 20(S)-Rg3 also exhibited suppressive effect on the MMP-9 gelatinolytic activity enhanced in the HaCat keratinocytes stimulated with tumor necrosis factor-α (TNF-α), one of the major proinflammatory cytokines. However, 20(S)-Rg3 was not able to modulate the NO level even in the presence of TNF-α. Taken together, anti-inflammatory and related antioxidative and MMP-9 inhibitory activities of 20(S)-Rg3, the major stereoisomeric form of ginsenoside Rg3, are confirmed in macrophage and keratinocyte cell lines.