ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Chen Huang,Ling-Jie Guo,Jia-Yi Li,Yue-Wen Chen,Bing Wu,Yi-Wei Liu,Chen-Zhi Zhang,Wei-Wei Lin,Ye Yang,Jin-Yuan Chen,Zhou-Jie Liu
The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10–250 nM (R2 = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.
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PHYTOTHERAPY RESEARCH
·
2026
·
IF 6.3
Yang Liu,Yan Lu,Weilian Bao,Haiyan Zhu,Guoqi Yuan,Linfeng Guo,Hong Li,Jiyang Li,Tong Wu,Xunlong Shi,Daofeng Chen
Secondary bacterial infection is a major cause of severe progression and death in viral pneumonia. The excessive recruitment of neutrophils accompanied by the formation of neutrophil extracellular traps (NETs) plays an important role in excessive inflammation caused by viral-bacterial co-infection, but effective pharmacological interventions remain lacking. Our study revealed that quercitrin (QTN), a flavonoid commonly found in traditional Chinese medicine and daily diets, significantly reduced mortality, recruitment of neutrophils, the production of inflammatory cytokines, and viral and bacterial loads in H1N1 influenza and methicillin-resistant Staphylococcus aureus (MRSA) coinfection-induced pneumonia in mice. Mechanistically, QTN down-regulates the interaction between Histone H3 and myeloperoxidase (MPO), thereby mitigating excessive inflammation induced by NETs. Our study demonstrates that inhibiting NETosis with QTN is one of the effective strategies for co-infection treatment.
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TALANTA
·
2026
·
IF 6.7
Luqin Lv,Yizhuo Zhang,Yaxin Fan,Beining Guo,Yijian Chen
Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.
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Microbial Drug Resistance
·
2020
·Vol. 26
·期 2
·100-109
·
IF 2.519
Chong-Fa Lai,Wei-Bin Xiao,Hua-Cheng Yan,Hui Yang,Lu-Xia Wang,Hui Guan,Qiu-ju Peng,Zhi-Rong Deng,Jian-Wen Chen,Lin-Kun An,Lei Shi
ZTW-41, an indolizinoquinoline-5,12-dione derivative, was investigated for antibacterial activity against Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). In our study, the MIC90s (minimum inhibitory concentrations) of ZTW-41 against MRSA (MRSA, n = 200), methicillin-sensitive S. aureus (MSSA, n = 100), Enterococcus faecalis (E. faecalis, n = 32), and Enterococcus faecium (E. faecium n = 32) were 0.25, 0.25, 0.125, and 8 μg/mL, respectively, whereas the MBC90s (minimum bactericidal concentrations) were 2, 1, 1, and >32 μg/mL, respectively. ZTW-41 maintained its potency at different pH levels (range 5–9) and in starting inoculum size up to 107 CFU/mL. The presence of human serum (25–75%) increased ZTW-41 MICs by two- to eightfold. Time-kill curves showed that ZTW-41 had bactericidal activity against MRSA, MSSA, and E. faecalis strains within 8 hours, and rebound growth occurred after 8 hours except at higher multiples of the MIC (4 × and 8 × ). In the acute toxicity study, no mortality or signs of toxicity was noted in mice after 14 days of observation at doses <50 mg/kg. ZTW-41 exhibited good selectivity indices (SIs) (SI = IC50/MIC90) ranging from 1.12 to 71.76 against clinical isolates, demonstrating excellent therapeutic selectivity in MRSA, MSSA, and E. faecalis strains. Moreover, the in vivo efficacy (effective dose [ED]50 = 6.59 mg/kg) of ZTW-41 was found comparable with vancomycin. Collectively, our favorable results supported ZTW-41 as a promising investigational candidate for treating drug-resistant bacteria infection.
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JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
·
2024
·
IF 3.1
Tingting Liu,Xiaomin Chen,Guanxuanzi Zhang,Jing Zhao,Qian Lu,Fang Wang,Hongxia Li,Bing Liu,Ping Zhu
Significant pharmacokinetic variation occurs in critically ill patients, leading to underexposure to antibiotics and poor prognosis. In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LC MS/MS) platform for the simultaneous quantification of 8 antibacterial and 2 antifungal drugs, which is optimally suited for clinically efficient, real-time therapeutic drug monitoring (TDM). Multiple reaction monitoring (MRM) mass spectrometry was used in this method, and samples were prepared via protein precipitation with methanol. Chromatographic separation was accomplished on a BGIU Column-U02 (2.1ｘ50 mm, 3 µm), with six stable isotopes and one analog as an internal standard. The overall turnaround time of the assay was 5 minutes. All the drugs tested (piperacillin, cefoperazone, meropenem, levofloxacin, moxifloxacin, daptomycin, linezolid, vancomycin, fluconazole and voriconazole) were linear in the test concentration range (r ≥ 0.9900), the accuracy was 95 %-111 %, the precision variation coefficient was greater than or equal to 10 %, the lower limit of quantitation was 0.31–7.51 mg/L, and the coefficient of variation of the matrix factor was less than 10 %. The recovery rates ranged from 85 % to 115 %, and the antibiotics were stable at 4°C and −20°C for 6 days, with an offset of greater than or equal to 15 %. This method was successfully applied to routine TDM in 252 elderly critically ill patients.
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Frontiers in Pharmacology
·
2025
·
IF 4.8
Shuai Dong,Xingyuan Bai,Bin Chen,Minzhe Fan,Qi Liu,Yubo Zhao,Linsen Li*,Dan Zhu
Background: Panzerina lanata (Lanata) is generally used to treat pustule infection in Inner Mongolia folk medicine and is called “the holy medicine for pustule.” However, the pharmacological mechanism of Lanata in treating pustule infection is still unclear.Aims: This study aimed to investigate the therapeutic effects of Lanata on skin infection and explore the underlying mechanisms.Methods: A skin wound methicillin-resistant Staphylococcus aureus (MRSA) infection mouse model was established to evaluate the healing effect of Lanata on infected wounds. In vitro assays were also conducted to determine the antibacterial activity of Lanata. Flow cytometry and immunohistochemistry were used to dynamically detect the number of neutrophils in the bone marrow, peripheral blood, and MRSA-infected wound. Protein expression in the infected wound skin was detected by a protein chip. Using an air pouch MRSA infection mouse model, the number of neutrophils, reactive oxygen species (ROS) level in neutrophils, and neutrophil extracellular trap (NET) formation were dynamically detected by flow cytometry and immunofluorescence. RNA-seq, RT-qPCR, flow cytometry, ELISA, and CXC chemokine receptor 2 (CXCR2) and P-selectin glycoprotein ligand-1 (PSGL-1) inhibitors were used to explore the mechanism of Lanata in regulating neutrophils.Results: In vitro assays showed that Lanata had no direct antibacterial activity. In skin wound MRSA-infected mouse, Lanata promoted the rapid migration of neutrophils from the bone marrow via peripheral blood to the wound site to eradicate MRSA in the acute stage of infection and accelerate wound healing. Skin protein chip analysis showed that Lanata upregulated CXCR2 and PSGL-1 protein levels in skin wounds. Furthermore, analysis using the air pouch MRSA infection mouse model found that Lanata not only promoted the rapid migration of neutrophils from peripheral blood to the air pouch but also enhanced the activation of neutrophils, including the increase of ROS and the release of NETs, and upregulated the expression of CXCR2, PSGL-1, and myeloperoxidase (MPO) in neutrophils. Inhibition of CXCR2 and MPO significantly attenuated the effect of Lanata on promoting migration and activation of neutrophils.Conclusion: Panzerina lanata resists MRSA infection by promoting migration and activation of neutrophils to rapidly eradicate MRSA.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
·
2025
·
IF 4.5
Jinyao Liu,Yingying Pang,Wenyan Li,Juanjuan Sun,Yujie He,Yonghong Guo,Jing Dong
Linezolid, an oxazolidinone antibiotic, is used in patients with liver or kidney disease. However, the effects and mechanisms of hepatic impairment or renal failure on the pharmacokinetics of linezolid and its metabolites (PNU-142586 and PNU-142300) remain unclear. We used carbon tetrachloride-induced impaired hepatic function and 5/6 nephrectomy-induced renal failure rat models to investigate linezolid and metabolite pharmacokinetics. Isolated primary rat hepatocytes were used to evaluate the impact of hepatic impairment or renal failure on linezolid metabolism. Uptake and efflux transport studies were also conducted. The influence of hepatic impairment or renal failure on the pharmacokinetics of linezolid and two metabolites did not differ between intragastric gavage and intravenous administration in rats. Linezolid did not accumulate in the brain, heart, lung, liver, kidney, and small intestinal tissues of the hepatic impairment or renal failure rats. And PNU-142300 did not accumulate in the liver or kidney tissue. Compared to the isolated normal rat hepatocytes, the in vitro hepatic clearance of linezolid in hepatic impairment and renal failure rat hepatocytes decreased by 61.3% and 44.1%, respectively. Organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, Na+-taurocholate co-transporting polypeptide (NTCP), organic anion transporter (OAT)1, OAT3, multidrug resistance-associated protein 2 (MRP2), or multidrug resistance protein 1 (MDR) did not mediate linezolid transport. Hepatic impairment primarily increases linezolid exposure through reduced hepatic metabolism, whereas renal failure increases both linezolid and two metabolites exposure through reduced hepatic metabolism and renal glomerular filtration. These findings guide adjusting the dose of linezolid in patients with hepatic and renal insufficiency.
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BIOMEDICAL CHROMATOGRAPHY
·
2025
·Vol. 39
·期 7
·e70107
·
IF 1.7
Jin-yao Liu,Ying-ying Pang,Piao-piao Wang,Wen-yan Li,Jing Dong
Linezolid is an oxazolidinone with potent antibacterial activity against multidrug resistant Gram-positive bacteria. This study aimed to develop and validate a sensitive, rapid and reliable high-performance liquid chromatography-photodiode array detector method for determining linezolid concentrations in the plasma and urine and to further characterize the pharmacokinetics of linezolid in rats. Chromatographic separation was performed using a SinoPak BEH C 18 column with isocratic elution (acetonitrile: water, 28:72, v/v). The retention times of linezolid and the internal standard are 3.30 and 5.20 min, respectively. The calibration curve for linezolid was linear over the concentration range of 0.25–50.0 μg/mL in rat plasma and 1.00–100.0 μg/mL in rat urine. Intra- and inter-assay precision values were below 9.00%, with accuracy ranging from 0.67% to 8.34%. Plasma samples were extracted using acetonitrile-mediated protein precipitation, and the recovery was > 93%. Following intragastric (63 mg/kg) and intravenous (25 mg/kg) administration of linezolid to rats, the area under the plasma concentration–time profile from time 0 to infinity were 224.5 and 86.4 μg·h/mL, total clearance were 0.29 and 0.30 L/h/kg, respectively. The cumulative urinary excretion rates of linezolid were 26.2%–27.5% for both administration routes. The presented method can certainly be used for routine analysis of linezolid in plasma and urine.
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MICROBIAL PATHOGENESIS
·
2025
·
IF 3.5
Tianmeng Li,Hong Shen,Peng Wang,Yu Wang,Jun Liu,Meibo He,Li Gu,Shuai Wang,Ming Wei
Enterococcus faecalis , a prevalent opportunistic pathogen, readily forms biofilms on surfaces, contributing to its virulence and antibiotic resistance. Previous studies have highlighted arachidonic acid (AA) as a promising antibiofilm agent. This investigation aimed to elucidate the antibiofilm activity of AA against linezolid-resistant E. faecalis and explore its potential molecular mechanisms. A total of 21 E. faecalis strains were included in this study: the standard strain ATCC 29212 and 20 linezolid-resistant clinical isolates. To determine the minimum inhibitory concentrations (MICs) of antibiotics and AA, the microbroth dilution method was performed. Checkerboard microdilution assays were also performed to assess the interaction between AA and linezolid. The crystal violet method was performed to evaluate the biofilm formation abilities of E. faecalis and the biofilm inhibition activities of different concentrations of AA (0.25 mM, 0.5 mM, and 1 mM). In addition, the expression levels of biofilm-related genes ( ebpA, ebpB, ebpC, gelE, ace, atlA, and esp ) were measured employing quantitative reverse transcription PCR (qRT-PCR). All clinical strains exhibited resistance to linezolid and tetracycline while remaining susceptible to other antibiotics, including penicillin, ampicillin, vancomycin, teicoplanin, and tigecycline. The MICs of AA against all 21 E. faecalis isolates were >1 mM. Synergy was observed in 75 % of strains (15/20), while additivity was observed in 25 % strains (5/20). All isolates displayed strong biofilm-forming abilities. AA significantly reduced biofilm formation with average inhibition rates of 68.16 %, 69.64 %, and 72.01 % at concentrations of 0.25 mM, 0.5 mM, and 1 mM, respectively ( P < 0.001). AA treatment resulted in decreased expression of ebpB, ebpC, ace, and atlA ( P < 0.001), while simultaneously increasing gelE expression ( P < 0.05). In conclusion, AA exhibited potent inhibitory effects on E. faecalis biofilm formation probably by influencing bacterial adhesion. The addition of AA can restore the susceptibility of linezolid against E. faecalis . These findings suggest that AA may serve as a potential therapeutic agent for preventing and treating E. faecalis infections, particularly those associated with biofilm formation.
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Virulence
·
2025
·
IF 5.4
Jian-Guo Li,Wei-Cheng Zhong,Long-Gen Zhong,Chao-Yan Niu,Ting-Yin Lu,Chuan-Jian Zhang,Jian Sun,Xiao-Ping Liao,Yu-Feng Zhou
Methicillin-resistant Staphylococcus aureus (MRSA) remains a major therapeutic challenge and poses a significant global health threat. Developing adjuvants to enhance the efficacy of existing antibiotics represents a promising strategy to address this issue. In this study, we evaluated auranofin as an adjuvant to potentiate the activity of linezolid against MRSA. Auranofin significantly increased MRSA susceptibility to linezolid, promoted intracellular linezolid accumulation, and suppressed the emergence of MRSA resistance to linezolid. Mechanistically, auranofin inhibited the Trx/TrxR system, inducing redox imbalance and reactive oxygen species (ROS) accumulation, which triggers DNA damage and transcriptional dysregulation. Auranofin synergized with linezolid to achieve dual inhibition of MRSA protein synthesis. Furthermore, auranofin downregulated the global regulator sarA, impaired SarA DNA-binding activity, and enhanced SarA phosphorylation, thereby attenuating SarA-mediated virulence factors (eg, adhesins and toxins) and biofilm formation. Importantly, auranofin fully restored anti-MRSA activity of linezolid in both Galleria mellonella and murine bacteremia models. Collectively, these findings identify auranofin as a promising adjuvant to linezolid and highlight its potential to improve therapeutic outcomes against invasive MRSA infections.
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