Chemical & Biomedical Imaging
·
2023
·
IF 0
Yan Zhang,Ning Xu,Chunyu Yan,Xuelian Zhou,Qinglong Qiao,Lu Miao,Zhaochao Xu
The aggregation of fusion in sarcoma (FUS) in the cytoplasm and nucleus is a pathological feature of Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD). Genetic mutations, abnormal protein synthesis, environmental stress, and aging have all been implicated as causative factors in this process. Salt ions are essential to many physiological processes in the body, and the imbalance of them is an important environmental stress factor in cells. However, their effect on liquid–liquid phase separation (LLPS) of FUS proteins in living cells is not well understood. Here, we map the various salt-induced LLPS of FUS in living cells by genetically coding and self-labeling FUS with organic dyes. The brightness and photostability of the dyes enable long-term imaging to track the mechanism of the assembly and disappearance of FUS phase separation. The FUS protein showed a better phase separation tendency under 0.3 M salt stimulation, and there was a large amount of FUS shuttling from the nucleus to the cytoplasm. At this concentration, various salt solutions displayed different effects on the phase separation of FUS protein, following the Hofmeister effects. We further observed that the assembly of FUS droplets underwent a process of rapid formation of small droplets, plateaus, and mutual fusion. Strikingly, The CsCl-stimulated FUS droplets were not completely reversible after washing, and some solid-like granules remained in the nucleus. Taken together, these results help broaden our understanding of the LLPS of FUS proteins in cellular stress responses.
研究领域:
神经退行性疾病
·
细胞生物学
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生物化学
·
分子生物学
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DOI
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
·
2026
·Vol. 123
·期 26
·e2610001123
·
IF 9.5
Dongyue Liu,Yu Li,Shuai Huang,Yufang Xu,Lei Sun,Wen Li,Cahir J. O’Kane,David C. Rubinsztein,Bingwei Lu,Shuangxi Li
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders characterized by motor neuron degeneration, leading to muscle weakness, atrophy, and cognitive impairments. A defining pathological hallmark of ALS/FTD is the cytosolic mislocalization and accumulation of TAR DNA-binding protein 43 (TDP-43), highlighting its critical role in ALS pathogenesis. However, the molecular mechanisms underlying TDP-43 proteostasis remain poorly understood. Through a genetic screening approach, we identify inositol-requiring enzyme 1 (IRE1), an endoplasmic reticulum-resident transmembrane protein, as a potent suppressor of TDP-43 protein levels. Furthermore, we show that ribosome-associated quality control (RQC) factors play a crucial role in regulating TDP-43 proteostasis and cellular toxicity. Activation of the RQC pathway prevents excessive accumulation of TDP-43 and associated toxicity. Mechanistically, our findings suggest that IRE1 regulates TDP-43 protein level by promoting the degradation of aberrant TDP-43 translation product through the RQC pathway. IRE1 acts canonically to enhance the transcription of the RQC core component Clbn/NEMF and noncanonically to physically interact with Clbn/NEMF, thereby ameliorating TDP-43-induced proteotoxicity. Moreover, ectopic expression or pharmacological activation of IRE1 alleviates TDP-43 pathology and restores cognitive function in the TDP-43 A315T ALS mouse models. Collectively, our study identifies a role for IRE1 in the translational quality control of TDP-43 and establishes its potential as a therapeutic target for ALS/FTD.
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DOI
高影响因子
THERAPEUTIC DRUG MONITORING
·
2015
·Vol. 37
·期 6
·776
·
IF 2.376
Zhang Zhi-qing,Dong Wei-chong,Yang Xiu-ling,Zhang Jin-feng,Jiang Xin-Hui,Jing Shao-jun,Yang Hong-le,Jiang Ye
Background: Free drug analysis is increasingly becoming popular in therapeutic drug monitoring (TDM). Centrifugal ultrafiltration (CF-UF) is the primary method to separate free drug from that of bound drug. However, the volume ratio of ultrafiltrate to sample solution ( V u / V s ) affects the accuracy of CF-UF, which highly depends on the different plasma conditions. Plasma protein concentrations in patients are different from those observed in healthy subjects, and there are also significant differences among patients with different diseases. Only very few studies have reported on the effect of protein concentration on the analysis methodology of free drug by CF-UF. Methods: In this study, valproic acid was used as the representative drug, and plasma samples with different albumin concentrations were analyzed by CF-UF and hollow fiber centrifugal ultrafiltration (HFCF-UF). Results: There was no significant difference of free drug concentrations by HFCF-UF and CF-UF when plasma albumin concentrations ranged 40–60 g/L. However, at low albumin concentrations (<40 g/L), a considerable difference was detected, and the difference was increased with the decrease of plasma albumin concentration. When the albumin concentration was as low as 10 g/L, the free drug concentration was 17.3 mcg/mL by CF-UF, whereas it was 10.2 mcg/mL by HFCF-UF. Conclusions: The accuracy of free drug measurement by CF-UF was albumin concentration dependent. However, such an effect was not observed when samples were prepared by HFCF-UF, which was more suitable for TDM of plasma samples from different patients. Therefore, this method could be readily applied to the measurement of free valproic acid plasma concentrations for TDM in patients.
研究领域:
药理学
·
临床医学
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DOI
PubMed
高影响因子
THERAPEUTIC DRUG MONITORING
·
2025
·
IF 2.4
Dong Zhonghua,Wang Peng,Cui Xueyan,Liu Jinjuan,Shi Haiyan,Yang Yilei,Xu Jingya,Yi Qiaoyan
Background: Perampanel (PER), a novel antiepileptic drug, exhibits high protein binding. Given the drug's susceptibility to various factors, including drug interactions, and the considerable variability in blood concentrations among individuals, solely measuring the total plasma concentration of PER may not provide comprehensive insights. This study aimed to establish an HPLC-FLD method to quantify both total and free PER in clinical samples. Methods: Analysis of total PER involved a straightforward sample preparation process involving plasma protein precipitation. Plasma samples were ultrafiltered to isolate the free portion of PER. Chromatographic separation was achieved on an InertSustain C18 column at a 1-mL/min flow rate using a gradient of acetonitrile and aqueous sodium acetate buffer at pH 4.4. Results: Calibration curves for total and free PER in plasma exhibited excellent linearity over the concentration ranges of 10–3000 and 0.5–100 ng/mL, respectively. The method was applied to analyze blood samples from patients with epilepsy quantitatively; the total concentration of perampanel in the plasma of the patients was in the range of 90.45–563.39 ng/mL, whereas the free concentration was in the range of 1.82–15.29 ng/mL. The plasma protein binding ratio of perampanel was normally between 97.09% and 99.29% and decreased in cases of hypoproteinemia. Conclusions: The method was rigorously validated for selectivity, accuracy, precision, and stability in accordance with established FDA and EMA guidelines. The developed method enables the rapid and accurate quantification of both total and free PER concentrations in the plasma of patients with epilepsy, offering technical support for its subsequent clinical application.
研究领域:
临床药理学
·
分析化学
·
神经病学
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产品关联
DOI
PubMed
高影响因子
THERAPEUTIC DRUG MONITORING
·
2024
·
IF 2.8
Fan Xudong,Guo Suhang,Zhang Ruoying,Cai Qingshan,Lang Yazhen,Huang Jinpeng,Chen Yuanyuan,Zhang Ying,Xu Yingying,Chen Meng,Yang Gaoyi,Cai Xinjun
Introduction: Linezolid, moxifloxacin, rifapentine, rifabutin, cycloserine, clofazimine, bedaquiline, levofloxacin, prothionamide, and ethionamide are commonly used second-line antituberculosis (anti-TB) drugs. To support therapeutic drug monitoring in regular clinical practice, the authors sought to develop a method based on ultra–high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) that would allow for the simultaneous quantification of multiple second-line anti-TB drugs in human serum. Methods: Analytes were extracted from human serum by protein precipitation. UHPLC-MS/MS was performed using a gradient at a flow rate of 0.3 mL/min, and each sample was taken for 7.5 minutes. The mass spectrometry scanning mode used was electrospray ionization with multiple reaction monitoring in the positive mode. Results: Validation showed that endogenous substances in the sample did not interfere with the assay, and the relationship between X and Y was highly linear, with a coefficient of determination (R 2 ) >0.9954 for each curve. The accuracy (85.0%–114.7%) and precision (intraday: 0.27%–9.32%; interday: 0.20%–7.66%) were less than 15.0%, and the internal standard-normalized matrix effects were consistent (coefficient of variation ≤4.40%). The analytes were stable in the final extract and human serum under various storage conditions (recovery: 87.0%–115.0%). The clinical applicability of the method was demonstrated by quantitative determination of analytes in serum samples obtained from patients with TB. Reproducibility of the drug concentrations measured in clinical samples was confirmed by incurred sample reanalysis. Conclusions: A simple and reliable analytical method was developed and validated for the simultaneous determination of 10 anti-TB drugs in human serum using UHPLC-MS/MS. Quantitation of anti-TB drugs in clinical samples confirmed that the assay is suitable for therapeutic drug monitoring in regular clinical practice.
研究领域:
药理学
·
临床化学
·
分析化学
·
结核病治疗
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DOI
PubMed
高影响因子
THERAPEUTIC DRUG MONITORING
·
2025
·
IF 2.4
Yu Hengyi,Hu Qun,Liu Lu,Xiang Dong,Yang Guangjie,Wang Yaqin,Wang Zhen,Liu Dong,Liu Aiguo,Gong Xuepeng
Background: Acute lymphoblastic leukemia (ALL) is the predominant malignancy in pediatric patients. As a crucial constituent of ALL chemotherapy, l -asparaginase is recognized as an integral element of treatment with a threshold concentration of 0.1 IU/mL used in treatment protocols. This study presents a novel liquid chromatography–tandem mass spectrometry method for evaluating plasma l -asparaginase activity in pediatric patients with ALL undergoing pegaspargase therapy. Methods: Initially, an enzyme incubation was conducted using 20 μL of plasma and 100 μL of l -asparagine (0.1 mol/L) at 37°C for 15 minutes. The reaction was stopped by adding sulfosalicylic acid and methanol. After the addition of isotope internal standard l -aspartic 13 C 4 acid, subsequent centrifugation, and dilution, the plasma samples underwent analysis by liquid chromatography–tandem mass spectrometry on a hydrophilic interaction liquid chromatography analytical column. A flow rate of 0.3 mL/min was used during isocratic elution using a mobile phase consisting of methanol–water (95:5, vol/vol) with 0.2% formic acid within a 5-minute run. Results: The calibration curves exhibited excellent linearity ranging from 0.1 to 15 IU/mL, with determination coefficients ( r 2 ) exceeding 0.99. The precision and accuracy ranged from 1% to 7% and from 93% to 110%, respectively. The relative recovery fell within the range of 98%–100%, and the internal standard–normalized matrix effect ranged from 95% to 101%. The stability was satisfactory across various conditions. Conclusions: This method was fully validated and successfully applied to quantify l -asparaginase activity in plasma samples of 15 children with ALL, enabling the monitoring of l -asparaginase activity with mass spectrometry.
研究领域:
临床化学
·
肿瘤学
·
儿科学
·
药代动力学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
THERAPEUTIC DRUG MONITORING
·
2025
·
IF 2.4
Ma Zhiyuan,Bai Mengru,Shen Shuying,Zhou Junshan,Dong Rong,Zhang Jiangjun,Weng Yayun,Li Li,Li Yongchen,Liu Dan,Yan Wei,Lin Nengming,Xia Jianmei
Background: Nirmatrelvir/ritonavir is licensed for the treatment of mild-to-moderate coronavirus disease (COVID-19) in patients at an increased risk of progression to severe disease. However, data on the real-world plasma exposure to nirmatrelvir/ritonavir remain limited, particularly in Chinese patients. This study aimed to assess the nirmatrelvir/ritonavir trough concentration (C trough ) and identify its critical factors in hospitalized Chinese patients treated with nirmatrelvir/ritonavir 300 mg/100 mg twice daily over a 5-day course. Methods: A high-performance liquid chromatography-tandem mass spectrometry assay was developed and validated to measure the nirmatrelvir/ritonavir C trough . Correlation analyses were performed to identify the variables influencing nirmatrelvir/ritonavir C trough . Results: Among the 110 patients, 100% had plasma concentrations above the antiviral in vitro 90% effective concentration. The median C trough of nirmatrelvir was 4.55 mcg/mL (15.6× 90% effective concentration), ranging from 0.65 to 12.44 mcg/mL. Nirmatrelvir C trough in normal and mild renal impairment cohorts were comparable (4.09 ± 1.97 mcg/mL and 4.57 ± 2.21 mcg/mL) but significantly increased in the moderate renal impairment cohort (6.41 ± 2.31 mcg/mL). Sex, age, and obesity were not significantly associated with nirmatrelvir exposure. Conclusions: Nirmatrelvir C trough was high in Chinese patients with COVID-19, and therapeutic drug monitoring should not be routinely recommended, except in patients with renal impairment.
研究领域:
药理学
·
传染病
·
临床研究
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产品关联
DOI
PubMed
高影响因子
CELL DEATH AND DIFFERENTIATION
·
2024
·
IF 13.7
Wu Yachen,Zheng Wenzhong,Xu Guofeng,Zhu Lijun,Li Zhiqiang,Chen Jincao,Wang Lianrong,Chen Shi
Sterol regulatory element binding transcription factors (SREBPs) play a crucial role in lipid homeostasis. They are processed and transported to the nucleus via COPII, where they induce the expression of lipogenic genes. COPII maintains the homeostasis of organelles and plays an essential role in the protein secretion pathways in eukaryotes. The formation of COPII begins at endoplasmic reticulum exit sites (ERES), and is regulated by SEC16A, which provides a platform for the assembly of COPII. However, there have been few studies on the changes in SEC16A protein levels. The repetitive expansion of the hexanucleotide sequence GGGGCC within the chromosome 9 open reading frame 72 (C9orf72) gene is a prevalent factor in the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we found that the absence of C9orf72 leads to a decrease in SEC16A protein levels, resulting in reduced localization of the guanine nucleotide exchange factor SEC12 at the ERES. Consequently, the small GTP binding protein SAR1 is unable to bind the endoplasmic reticulum normally, impairing the assembly of COPII. Ultimately, the disruption of SREBPs transport decreases de novo lipogenesis. These results suggest that C9orf72 acts as a novel role in regulating lipid homeostasis and may serve as a potential therapeutic target for obesity.
研究领域:
分子生物学
·
脂质代谢
·
神经退行性疾病
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产品关联
DOI
PubMed
高影响因子
EXPERIMENTAL NEUROLOGY
·
2022
·
IF 5.62
Shusheng Wu,Yuye Yin,Longfei Du
As is the case with neurodegenerative diseases , abnormal accumulation of aggregated proteins in neurons and glial are also known to implicate in the pathogenesis of ischemic stroke . However, the potential role of protein aggregates in brain ischemia remains largely unknown. Fused in Sarcoma (FUS) protein has a vital role in RNA metabolism and regulating cellular homeostasis . FUS pathology has been demonstrated in the formation of toxic aggregates and critically affecting cell viability in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but whether this also applies to neurological injury following cerebral ischemia is unclear. Herein, we demonstrated a critical role of aggregated FUS in astrocyte activation caused by cerebral ischemia and a possible underlying molecular mechanism. Cerebral ischemic injury significantly induced the formation of cytoplasmic FUS aggregates in reactive astrocytes and injured neurons, thereby aggravating neurofunctional damages and worsening stroke outcomes. Further analysis revealed that extranuclear aggregation of FUS in astrocytes was involved in the induction of excessive autophagy, which contributes to autophagic cell injury or death. In conclusion, our results reveal the important contribution of FUS aggregates in promoting astrocyte activation in stroke pathology independent of its transcriptional regulation activity. We thus propose that aggregation of FUS is an important pathological process in ischemic stroke and targeting FUS aggregates might be of unique therapeutic value in the development of future treatment strategies for ischemic stroke.
研究领域:
神经科学
·
病理学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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