In Situ Track-Generated DNA Walker for AND-Gate Logic Imaging of Telomerase and Flap Endonuclease 1 Activities in Living Cells.

In situ monitoring of the actions of correlated enzymes in living cells is crucial for expanding our understanding of disease progression and evaluating drug efficacy. However, due to the diverse functions of different enzymes, currently available methods for comprehensive analysis of these events are limited. Here, we present an in situ track-generated DNA walker for AND-gate logic imaging of telomerase (TE) and flap endonuclease 1 (FEN1) activities in live cells. TE is in charge of generating the tracks for the walking strands by extending the TE primer on a gold nanoparticle, while FEN1 is responsible for recognizing the overlapping structure formed by the walking strands and the tracks and then cleaving the fluorescent reporter to produce signals. By utilizing the DNA walker, we successfully determined the expression levels and activities of TE and FEN1 in various cancer cell lines, offering promising prospects for screening inhibitors and investigating the biomolecular mechanisms of diseases.

A DNA Nano Firework for Imaging and Inhibitor Screening of Flap Endonuclease 1 in Living Cells.

In situ observation of changes in the activity of marker proteins in living cells is crucial for both biomarker-based disease diagnosis and drug screening. Flap endonuclease 1 (FEN1) has been recognized as a broad-spectrum cancer biomarker and therapeutic target. However, simple and reliable methods for in situ studying the FEN1 activity changes in living cells are limited. Here, we introduce a nano firework as a fluorescent sensor to sense and report FEN1 activity changes in living cells through FEN1 recognizing the substrates on the surface of the nano firework to release and restore the fluorescence of the prequenched fluorophores. We verified the high selectivity, anti-interference ability, stability, and quantitative performance of the nano firework in tubes and living cells, respectively. A series of controlled experiments have demonstrated that the nano firework could accurately report changes in FEN1 activity in different cells, enabling "sensors in, results out" in the manner of simple addition to the cell culture medium. Using an in silico molecular docking study and experiments, we also explored the ability of the nano firework for rapid screening of FEN1 inhibitors and found two new candidate compounds myricetrin and neoisoliquritin, which could be used as FEN1 inhibitors for further research. These performances of the nano firework suggest that it can be used in high-throughput screening applications, providing a promising tool for biomarker-based new drug discovery.

Development and validation of a highly sensitive and selective LC-MS/MS method for the determination of 15-hydroxylubiprostone in human plasma: application to a pharmacokinetic study in healthy Chinese volunteers.

Lubiprostone, a derivative of prostaglandin E1, is the first chemical-type constipation treatment approved by FDA. Lubiprostone has low systemic exposure after oral administration. Therefore, it is recommended that 15-hydroxylubiprostone, which is a dominant active metabolite of lubiprostone, be used as the pharmacokinetic evaluation indicator. Due to the microdosage of the lubiprostone capsules, it is difficult to develop a highly sensitive bioanalytical method for 15-hydroxylubiprostone.In this study, a highly sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been established and fully validated for the quantification of 15-hydroxylubiprostone in human plasma, and the validated bioanalytical method has been applied to a pharmacokinetic study of lubiprostone capsules successfully.The pharmacokinetics of 15-hydroxylubiprostone were observed after fed administration in healthy Chinese volunteers. The Cmax and AUC0-t were 75.8 ± 57.6 pg/mL and 222 ± 68.0 pg·h/mL for 15-hydroxylubiprostone.This study investigated the pharmacokinetic properties of 15-hydroxylubiprostone under fed conditions in healthy Chinese volunteers and would provide clinical guidance for the application and further development of lubiprostone capsules.

Multiplex Visualized Closed-Tube PCR with Hamming Distance 2 Code for 15 HPV Subtype Typing.

Cervical cancer is the fourth leading cause of death in women, especially in developing countries. Specific and economic methodologies for HPV typing are crucial in cancer diagnosis and further disease control. However, routine assays based on real-time polymerase chain reaction (qPCR) or DNA-chip hybridization are either incapable of offering detailed subtype information or involve tedious open-tube operations with the risk of cross-contamination from PCR amplicons. Herein, we proposed a multiplex visualized closed-tube PCR (Multi-Vision) for HPV typing. Using gold nanoparticle probes (AuNPs) as a color change indicator combined with a Hamming distance 2 coding scheme, 13 high-risk HPVs and two subtypes associated with high-incidence benign lesions were successfully typed by performing six closed-tube PCRs. The assay demonstrates high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 100 to 103 copies per reaction) and enables highly sensitive detection of as low as 0.5 copies/µL. Further, 105 clinical samples were successfully analyzed using our method with a high concordance rate of 99.05% (104/105) compared to a HPV typing kit. The inconsistent sample was confirmed by sequencing to be consistent with the typing results determined by our method, indicating that Multi-Vision could be a useful tool for HPV detection, especially in resource-limited regions.

Flap Endonuclease 1-Assisted DNA Walkers for Sensitively and Specifically Sensing ctDNAs.

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

Point-of-care DNA testing by automatically and sequentially performing extraction, amplification and identification in a closed-type cassette.

Nucleic acid detection is important for clinical diagnostics; however, it is challenging to perform genetic testing at the point-of-care due to the tedious steps involved in DNA extraction and the risk of cross-contamination from amplicons. To achieve a fully-automated and contamination-free nucleic acid detection, we propose a closed-type cassette system which enables the following steps to be operated automatically and sequentially: sample preparation based on magnetic beads, target amplification using multiplex polymerase chain reaction, and colorimetric detection of amplicons using a serial invasive reaction coupled with the aggregation of gold nanoparticle probes. The cassette was designed to be round and closed, and 10 targets in a sample could be simultaneously detected by the naked eye or using a spectrophotometer in the system. In addition, a cassette-driven device was fabricated to transfer reagents between wells, to control the temperature of each reaction, and to sense the colour in the detection wells. The cassette system was sensitive enough to detect 10 genotypes at 5 single nucleotide polymorphism sites related to the anticoagulant's usage, by using a 0.5 µL blood sample. The accuracy of the system was evaluated by detecting 12 whole blood samples, and the results obtained were consistent with those obtained using pyrosequencing. The cassette is airtight and the whole system is fully automatic; the only manual operation is the addition of the sample to the cassette, performing point-of-care genetic testing in a sample-in/answer-out way.

Predicting Pharmacokinetics Variation of Faropenem Using a Pharmacometabonomic Approach.

Individual variation in pharmacokinetics of faropenem is significant. We attempted to predict drug response of faropenem using a pharmacometabonomic approach. Metabolic profiling was performed on predose plasma samples from 36 healthy volunteers by gas chromatography-mass spectrometry (GC/MS), with 204 endogenous metabolites detected. Plasma concentration was measured after drug administration, using high pressure liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the pharmacokinetic parameters were calculated. Then a two-stage partial least squares strategy was employed to screen potential biomarkers and predict the pharmacokinetic parameters of faropenem. The results showed a good prediction of AUC and Cmax with the screened biomarkers, and metabolites such as valine, proline, aspartic acid, gluconic acid, glucuronic acid, and 2-ketoisocaproic acid were indicated as candidate biomarkers. Finally, we explored the mechanism of individual variation by pathway enrichment analysis, and it suggested that organic anion transporter 1 (OAT1) and 3 (OAT3) might be responsible for individual variation of faropenem, and this hypothesis was verified by an experiment in rats.

Integration analysis of metabolites and single nucleotide polymorphisms improves the prediction of drug response of celecoxib.

INTRODUCTION: Pharmacogenetics and pharmacometabolomics are the common methods for personalized medicine, either genetic or metabolic biomarkers have limited predictive power for drug response. OBJECTIVES: In order to better predict drug response, the study attempted to integrate genetic and metabolic biomarkers for drug pharmacokinetics prediction. METHODS: The study chose celecoxib as study object, the pharmacokinetic behavior of celecoxib was assessed in 48 healthy volunteers based on UPLC-MS/MS platform, and celecoxib related single nucleotide polymorphisms (SNPs) were also detected. Three mathematic models were constructed for celecoxib pharmacokinetics prediction, the first one was mainly based on celecoxib-related SNPs; the second was based on the metabolites selected from a pharmacometabolomic analysis by using GC-MS/MS method, the last model was based on the combination of the celecoxib-related SNPs and metabolites above. RESULTS: The result proved that the last model showed an improved prediction power, the integration model could explain 71.0% AUC variation and predict 62.3% AUC variation. To facilitate clinical application, ten potential celecoxib-related biomarkers were further screened, which could explain 68.3% and predict 54.6% AUC variation, the predicted AUC was well correlated with the measured values (r = 0.838). CONCLUSION: This study provides a new route for personalized medicine, the integration of genetic and metabolic biomarkers can predict drug response with a higher accuracy.

Sequence-encoded quantitative invader assay enables highly sensitive hepatitis B virus DNA quantification in a single tube without the use of a calibration curve.

Accurately quantifying hepatitis B virus DNA (HBV-DNA) in serum is important in dynamic monitoring and prognosis evaluation for patients with hepatitis B. Routine assays based on real-time polymerase chain reaction (qPCR) for HBV-DNA quantification usually require laborious calibration curves and may bring bias from the biological samples. To enable absolute quantification of HBV-DNA in a single tube, we described a modification of the conventional Q-Invader assay by separately encoding targeted DNA and artificially designed internal quantitative-standard DNA (QS-DNA) at the flaps of the corresponding downstream probes. Quantification of targeted HBV-DNA was readily achieved by the difference in the quantification cycle value (Ct) between itself and QS-DNA. Furthermore, spiked-in QS-DNA before DNA extraction allowed errors caused by DNA extraction to be corrected. Two different gene regions covering eight genotypes were encoded with the same flap to avoid false-negative results. The method demonstrates a high sensitivity, which enables accurate detection of as low as 2 copies of the HBV-DNA plasmid or 20 IU mL-1 HBV-DNA in serum in a single tube. Successful quantification of 50 clinical samples indicates that our method is cost-effective, labor-saving and reproducible, and promising for the ultra-sensitive quantification analysis of many types of pathogens other than HBV.

Highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA.

Detection of EGFR mutations in circulating cell-free DNA (cfDNA) is beneficial to monitor the therapeutic effect, tumor progression, and drug resistance in real time. However, it requires that the mutation detection method has the ability to quantify the mutation abundance accurately. Although the next-generation sequencing (NGS) and digital PCR showed high sensitivity for quantifying mutations in cfDNA, the use of expensive equipment and the high-cost hampered their applications in the clinic. Herein, we propose a highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA (termed as qPCR-Invader). The mutation abundance can be quantified by using the difference of Ct values between mutant and wild-type targets without the need of making a standard curve. The method can quantify a mutation level as lower as 0.1% (10 copies/tube). Thirty-six tissue samples from non-small-cell lung cancer (NSCLC) patients were detected by our method and 14/36 tissues gave EGFR L858R mutation-positive results, whereas ARMS-PCR just identified 12 of L858R mutant samples. The two inconsistent samples were confirmed as L858R mutant by pyrophosphorolysis-activated polymerization method, indicating that qPCR-Invader is more sensitive than ARMS-PCR for mutation detection. The L858R mutation abundances of 19 cfDNA samples detected by qPCR-Invader were close to that from NGS, indicating our method can precisely quantify mutation abundance in cfDNA. The qPCR-Invader just needs a common real-time PCR device to accomplish quantification of EGFR mutations, and the fluorescence probes are universal for any target detection. Therefore, it could be used in most laboratories to analyze mutations in cfDNA. Graphical abstract ᅟ.

Bacterial communities under long-term conventional and transgenic cotton farming systems using V3-V5 and V5-V9 of 16s rDNA.

Understanding the community structure of soil microbes is required to evaluate the potential effects of genetically modified (GM) plants on ecological environments. Bacterial communities in soil planted with conventional cotton (CC) and transgenic cultivar (TC) in a natural ecosystem for three years were characterized by 454 pyrosequencing of the V3-V5 and V5-V9 regions of 16S rDNA from June to September 2013. V3-V5 and V5-V9 regions yielded a total of 12,848 and 10,541 OTUs, respectively. The V5-V9 amplicon was additionally used to detect phyla that were poorly sequenced by V3-V5 (such as Chlamydiae, Crenarchaeota and Archaea). Among the species detected by each primer pair, 46% of the species identified from V3-V5 and 60% of those identified from V5-V9 were detected by both primer pairs. Although distinct bacterial compositions existed between the two amplified regions, statistical analysis revealed no significant difference in the diversity indexes or phylogenetic patterns in TC versus compared to those in the CC control. Further, clustering analysis in both regions indicated that there was no unambiguous aggregation in TC compared to that in CC control. Of all 26 phyla detected by both regions, each region detected 2 distinct phyla exhibiting significant variations in abundance. The species unique to each treatment field accounted for less than 27% of all species and were rare taxa (abundance < 0.15%). However, a small fraction of diagnostic taxa with specific ecological functions differed significantly between TC and CC. These differences were not driven by any obvious environmental factors. The results established a comprehensive inventory of the bacterial communities associated with GM plants and indicated that transgenic cotton may not significantly affect soil microorganisms compared with conventional cotton over a three-year period. Furthermore, diagnostic taxa were provided for monitoring the perturbation in soil, but further verification in future studies is required.

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

A Pharmacometabonomic Approach To Predicting Metabolic Phenotypes and Pharmacokinetic Parameters of Atorvastatin in Healthy Volunteers.

Genetic polymorphism and environment each influence individual variability in drug metabolism and disposition. It is preferable to predict such variability, which may affect drug efficacy and toxicity, before drug administration. We examined individual differences in the pharmacokinetics of atorvastatin by applying gas chromatography-mass spectrometry-based metabolic profiling to predose plasma samples from 48 healthy volunteers. We determined the level of atorvastatin in plasma using liquid chromatography-tandem mass spectrometry. With the endogenous molecules, which showed a good correlation with pharmacokinetic parameters, a refined partial least-squares model was calculated based on predose data from a training set of 36 individuals and exhibited good predictive capability for the other 12 individuals in the prediction set. In addition, the model was successfully used to predictively classify individual pharmacokinetic responses into subgroups. Metabolites such as tryptophan, alanine, arachidonic acid, 2-hydroxybutyric acid, cholesterol, and isoleucine were indicated as candidate markers for predicting by showing better predictive capability for explaining individual differences than a conventional physiological index. These results suggest that a pharmacometabonomic approach offers the potential to predict individual differences in pharmacokinetics and therefore to facilitate individualized drug therapy.

Progress in multiplex loop-mediated isothermal amplification technology.

Loop-mediated isothermal amplification (LAMP) has been widely applied in nucleic acid diagnostics due to its high sensitivity and specificity, high speed and low requirement of equipment. In order to fully leverage these merits, achieve high efficiency and reliability in diagnostics, and expand the applicable fields while keeping low reagent cost, multiplex LAMP technology has been extensively explored in recent years. Common methods for LAMP products detection are mostly based on the double-stranded DNA amplicons or byproducts from the polymerization reaction, so they can only identify the occurrence of amplification reaction but not the origins or specificity of the products. To achieve specific LAMP products detection, researchers developed various multiplex methods by improving the conventional LAMP technology or coupling LAMP with other assays. However, the interference and/or the different amplification efficiencies among different primer sets often lead to biased amplification and thus limited multiplexing level. We here defined these methods as narrow-sensed multiplex LAMP. The research on miniaturized amplification technology which is booming in recent years has given rise to the novel general-sensed multiplex LAMP technology that breaks this limitation by its capability to perform highly parallel and miniaturized simplex reactions in independent compartments. Methods of this type have additional benefits such as lower reagent cost, higher level of automation, lower risk of cross-contamination and better suitability for on-site detection of multiple targets. In this review, we summarize the recent research progress in multiplex LAMP technology from the following aspects: the principle and design of narrow-sensed LAMP and its amplification optimization, the general-sensed LAMP, and the various applications of all multiplex LAMP technologies in diagnostics.

Development and validation of pharmacokinetics assays for a novel HER2-targeting antibody-drug conjugate (SHR-A1201): Application to its dose-escalation pharmacokinetic study.

Approximately 25% of breast cancer patients with HER2 overexpression tend to have a high risk of disease progression and death. Various HER2-targeting therapies have been approved for treatment. Recently, a novel antibody-drug conjugate, SHR-A1201, is being researched and developed. For the pharmacokinetic study of SHR-A1201, suitable bioanalytical methods are needed for quantifying unconjugated cytotoxin, cytotoxin-conjugated antibodies and total antibodies. In this research, bioanalytical methods involving a highly sensitive LC-MS/MS assay for unconjugated cytotoxic payload DM1 in human plasma, ELISA strategies for DM1-conjugated trastuzumab and total trastuzumab in human serum were developed, validated and successfully applied to a phase I dose-escalation pharmacokinetic study of SHR-A1201. The pharmacokinetic properties and exposure-to-dose proportionality was evaluated for SHR-A1201. According to the bioanalytical method validation guidance, the bioanalytical methods were fully validated and the validation results met the acceptance criteria. The nonspecific binding of DM1 and dimer was avoided for the LC-MS/MS assay. In the dose-escalation pharmacokinetic study of SHR-A1201, a potential dose-proportional pharmacokinetics was observed over the dose from 1.2 mg/kg to 4.8 mg/kg. The validated bioanalytical strategies are robust and reproducible and these bioanalytical methods will contribute to better understanding of the pharmacokinetic properties of SHR-A1201.

The investigation of connection between anticoagulant therapy and vitamin K homologues in human determined by LC-MS/MS.

Aim: Differences are existed in the bioactivity among various vitamin K (VK) forms. To investigate the correlation between clinical parameters of initial anticoagulation and plasma levels of VK1 and VK2 (MK-4 and MK-7), it was necessary to establish a quantitative method for simultaneous determination.Materials & methods: Plasma samples in cardiovascular patients were extracted by cyclohexane and analyzed using a C18 column. Baseline concentrations of VK1, MK-4 and MK-7 were 0.98 ± 0.52 ng/ml, 0.45 ± 0.13 ng/ml and 0.65 ± 0.31 ng/ml, respectively. The concentrations of MK-7 and total VKs were significantly relevant to INR0, respectively (p = 0.010 and p = 0.048, respectively).Conclusion: Thus, when adjusting anticoagulation dosage, concentrations of various VK homologues might be considered.

[Box: see text].

LC-MS/MS method for dual-ligand peptide-drug CBP-1018 and its deconjugated payload MMAE including sample stabilization strategy for its MC-Val-Cit-PABC linker.

Recently, peptide-drug conjugate (PDC) has become the most promising conjugated drug for tumor therapy after antibody-drug conjugate due to stronger tumor penetration capacity and lower immunogenicity. CBP-1018 was a PDC with dual-ligand conjugated to MMAE via a cleavable linker (MC-Val-Cit-PABC) that can be lysed by cathepsins B. In this study, two specific LC-MS/MS methods were developed and validated for the determination of CBP-1018 and its metabolite MMAE in human plasma. To prevent the cleavable MC-Val-Cit-PABC linker from degradation, a protease inhibitor (cOmplete solution) was added to the pre-cooled vacuum tubes and the separated plasma samples. The assays involved the pretreatment of CBP-1018 by protein precipitation with H2O-ACN (1:9, v/v) and the extraction of MMAE by liquid-liquid extraction with ethyl acetate under alkaline condition to eliminate the interference of CBP-1018 on MMAE. The two analytes showed good linearities over the calibration ranges (R2 ≥ 9980). Both accuracy and precision met the acceptance criteria. The validated methods were successfully applied to the phase I dose-escalation study of CBP-1018 injection in Chinese patients with solid tumors to evaluate the pharmacokinetic properties of CBP-1018 and MMAE. The results showed that CBP-1018 was eliminated immediately after injection and MMAE reached the maximum exposure at approximately 2 h after infusion. The maximum concentration of MMAE did not exceed 20.0 ng/mL, suggesting that the off-target toxicity of CBP-1018 injection was controllable.

FEN1-assisted DNA logic amplifier circuit for fast and compact DNA computing.

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

Exchange Bias Modulated by Antiferromagnetic Spin-Flop Transition in 2D Van der Waals Heterostructures.

Exchange bias is extensively studied and widely utilized in spintronic devices, such as spin valves and magnetic tunnel junctions. 2D van der Waals (vdW) magnets, with high-quality interfaces in heterostructures, provide an excellent platform for investigating the exchange bias effect. To date, intrinsic modulation of exchange bias, for instance, via precise manipulation of the magnetic phases of the antiferromagnetic layer, is yet to be fully reached, owing partly to the large exchange fields of traditional bulk antiferromagnets. Herein, motivated by the low-field spin-flop transition of a 2D antiferromagnet, CrPS4, exchange bias is explored by modulating the antiferromagnetic spin-flop phase transition in all-vdW magnetic heterostructures. The results demonstrate that undergoing the spin-flop transition during the field cooling process, the A-type antiferromagnetic ground state of CrPS4 turns into a canted antiferromagnetic one, therefore, it reduces the interfacial magnetic coupling and suppresses the exchange bias. Via conducting different cooling fields, one can select the exchange bias effect switching among the "ON", "depressed", and "OFF" states determined by the spin flop of CrPS4. This work provides an approach to intrinsically modulate the exchange bias in all-vdW heterostructures and paves new avenues to design and manipulate 2D spintronic devices.

Rivaroxaban for Thromboembolism Prophylaxis in Patients with Nephrotic Syndrome: A Single-Arm, Prospective Study.

Introduction: Thromboembolism is a recognized complication of nephrotic syndrome (NS). Evidence supporting the use of rivaroxaban to prevent NS-related thrombosis is limited and controversial. This study aimed to explore the impact of NS on rivaroxaban pharmacokinetics and to collect observational data on the efficacy and safety of rivaroxaban as primary thromboprophylaxis in patients with NS. Methods: This prospective study analyzed 141 patients with NS who received rivaroxaban (10 mg/day) for thromboprophylaxis. High-performance liquid chromatography-tandem mass spectrometry was used to measure the trough and peak plasma concentrations (Ctrough and Cmax) of rivaroxaban. The influence of clinical and genetic factors on these concentrations was examined using multivariate logistic regression. Results: The median Cmax and Ctrough were 68.5 ng/mL (interquartile range [IQR], 31.7-105.5 ng/mL) and 4.4 ng/mL (IQR, 1.2-11.9 ng/mL), respectively. The incidence of thromboembolic events (TEs) was 12.8%, while that of bleeding events was 14.2%, although all were classified as minor. Albumin level was the most significant factor affecting Cmax (ρ = 0.55; p < 0.001) and was also significantly associated with TEs (0.81; 0.71-0.91 per 1.0 g/dL increase; p = 0.001) and bleeding risks (1.11; 1.03-1.19 per 1.0 g/dL increase; p = 0.008). Single nucleotide polymorphisms in the ABCB1 gene significantly influenced Ctrough but were not associated with clinical outcomes. Conclusion: Hypoalbuminemia significantly affects the pharmacokinetics of rivaroxaban in NS patients. A dose-adjustment strategy based on rivaroxaban concentrations, accounting for variable albumin levels, may improve the safety and efficacy of thromboprophylaxis in this population.