Susceptibility of hypertensive individuals to acute blood pressure increases in response to personal-level environmental temperature decrease.

BACKGROUND: Environmental temperature is negatively associated with blood pressure (BP), and hypertension may exacerbate this association. The aim of this study is to investigate whether hypertensive individuals are more susceptible to acute BP increases following temperature decrease than non-hypertensive individuals. METHODS: The study panel consisted of 126 hypertensive and 125 non-hypertensive (n = 251) elderly participants who completed 940 clinical visits during the winter of 2016 and summer of 2017 in Beijing, China. Personal-level environmental temperature (PET) was continuously monitored for each participant with a portable sensor platform. We associated systolic BP (SBP) and diastolic BP (DBP) with the average PET over 24 h before clinical visits using linear mixed-effects models and explored hourly lag patterns for the associations using distributed lag models. RESULTS: We found that per 1 °C decrease in PET, hypertensive individuals showed an average (95 % confidence interval) increase of 0.96 (0.72, 1.19) and 0.28 (0.13, 0.42) mmHg for SBP and DBP, respectively; and non-hypertensive participants showed significantly smaller increases of 0.28 (0.03, 0.53) mmHg SBP and 0.14 (-0.01, 0.30) mmHg DBP. A lag pattern analysis showed that for hypertensive individuals, the increases in SBP and DBP were greatest following lag 1 h PET decrease and gradually attenuated up to lag 10 h exposure. No significant BP change was observed in non-hypertensive individuals associated with lag 1-24 h PET exposure. The enhanced increase in PET-associated BP in hypertensive participants (i.e., susceptibility) was more significant in winter than in summer. CONCLUSIONS: We found that a decrease in environmental temperature was associated with acute BP increases and these associations diminished over time, disappearing after approximately 10 hours. This implies that any intervention measures to prevent BP increases due to temperature drop should be implemented as soon as possible. Such timely interventions are particularly needed for hypertensive individuals especially during the cold season due to their increased susceptibility.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation.

Polydopamine (pDA) is a valuable material with wide-ranging potential applications. However, the complex and debated nature of dopamine polymerization complicates our understanding. Specifically, the impact of foreign substances, especially proteins, on pDA formation adds an additional layer of subtlety and complexity. This study delves into specific surface features of proteins that predominantly shape their impact on dopamine polymerization. Notably, the biotin-binding site emerges as a critical region responsible for the pronounced inhibitory effect of avidin and neutravidin on the dopamine polymerization process. The binding of biotin successfully mitigates these inhibitory effects. Moreover, several nucleases demonstrated a significant hindrance to pDA formation, with their impact substantially alleviated through the introduction of DNA. It is speculated that hydrogen bonding, electrostatic, cation-π, and/or hydrophobic interactions may underlie the binding between protein surfaces and diverse oligomeric intermediates formed by the oxidation products of dopamine. Additionally, we observed a noteworthy blocking effect on the dopamine polymerization reaction induced by erythropoietin (EPO), a glycoprotein hormone known for its role in stimulating red blood cell production and demonstrating neuroprotective effects. The inhibitory influence of EPO persisted even after deglycosylation. These findings not only advance our comprehension of the mechanisms underlying dopamine polymerization but also provide strategic insights for manipulating the reaction to tailor desired biomaterials.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Exploring the therapeutic potential of recombinant human lysozyme: a review on wound management system with antibacterial.

Lysozyme, a natural antibacterial enzyme protein, possesses the ability to dissolve the cell walls of Gram-positive bacteria, demonstrating broad-spectrum antibacterial activity. Despite its significant potential in treating wound infections and promoting wound healing, its widespread clinical application has yet to be realized. Current research is primarily focused on carrier-based delivery systems for lysozyme. In this review, we discuss four delivery systems that can be employed for lysozyme in wound healing treatment, specifically hydrogels, nanofilms, electrospun fibrous membranes, and modified-lysozyme composite systems. These systems not only enhance the stability of lysozyme but also enable its controlled and sustained release at wound sites, potentially overcoming some of the challenges associated with its direct application. Lastly, we delve into the perspectives and challenges related to the use of these delivery systems, hoping to spur further research and innovation in this promising field.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

Visualization and Comparison of the Level of Apurinic/Apyrimidinic Endonuclease 1 in Live Normal/Cancerous and Neuron Cells with a Fluorescent Nanoprobe.

As a major apurinic/apyrimidinic endonuclease and a redox signaling protein in human cells, APE1 plays a crucial role in cellular function and survival. The relationship between alterations of APE1 expression and subcellular localization and the initiation, development and treatment of various cancers has received extensive attention. However, comparing the in-vivo activity of APE1 in normal and cancerous breast live cells remains challenging due to the low efficiency of commonly used liposome transfection methods in delivering DNA substrate probes into human normal breast epithelial cells (MCF-10A). In this work, we develop a DNA/RNA hybrid-based small magnetic fluorescent nanoprobe (25 ± 3 nm) that can be taken up by various live cells under magnetic transfection. The D0/R-nanoprobe demonstrates an outstanding specificity toward APE1 and strong resistance to the cellular background interference. Using this nanoprobe, we are not only able to visualize the intracellular activity of APE1 in breast ductal carcinoma (MCF-7) live cells, but also demonstrate the APE1 activity in MCF-10A live cells for the first time. The method is then extended to observe the changes in APE1 levels in highly metabolically active neuroendocrine cells under normal conditions and severe attacks by reactive oxygen species in real-time. The fluorescent nanoprobe provides a useful tool for studying the dynamic changes of intracellular APE1 in normal or cancerous live cells. It also displays the potential for visible and controllable release of miRNA drugs within live cells for therapeutic purposes.

Modular Oxidation of Cytosine Modifications and Their Application in Direct and Quantitative Sequencing of 5-Hydroxymethylcytosine.

Selective, efficient, and controllable oxidation of cytosine modifications is valuable for epigenetic analyses, yet only limited progress has been made. Here, we present two modular chemical oxidation reactions: conversion of 5-hydroxymethylcytosine (5hmC) into 5-formylcytosine (5fC) using 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate (ACT+BF4-) and further transformation of 5fC into 5-carboxycytosine (5caC) through Pinnick oxidation. Both reactions are mild and efficient on double-stranded DNA. We integrated these two oxidations with borane reduction to develop chemical-assisted pyridine borane sequencing plus (CAPS+), for direct and quantitative mapping of 5hmC. Compared with CAPS, CAPS+ improved the conversion rate and false-positive rate. We applied CAPS+ to mouse embryonic stem cells, human normal brain, and glioblastoma DNA samples and demonstrated its superior sensitivity in analyzing the hydroxymethylome.

Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and "interactomes" of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH2 ). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.

A homogeneous fluorescence assay for rapid and sensitive quantification of the global level of abasic sites in genomic DNA.

The apurinic/apyrimidinic (AP) sites are frequent DNA lesions in genomic DNA (gDNA). Here we report a facile approach for rapid quantification of the AP sites in gDNA with high selectivity and sensitivity. With the assistance of T4 pyrimidine dimer glycosylase, we covalently labeled the AP sites with 5'-hydroxylamine-modified oligonucleotide strand with high chemical selectivity against to naturally occurring formylated-bases, such as 5-formylcytosine and 5-formyluracil. Next, we sequentially removed the excessive labeling strands and triggered a signal amplification reaction with the labeled strands in a homogeneous system by flexible variation of the 3' or 5' terminal bases of an assistant strand and a fluorescent probe in the presence of a versatile exonuclease (lambda exonuclease). The detection of AP sites in gDNA was realized with an input of gDNA less than 500 ng and a limit of detection down to 0.2 fmol. The method enabled quantification of AP sites in gDNA from both normal cells and cells exposed to external damaging agents, showing the variation of AP sites level along with damaging and repair processes. The work has also provided a useful strategy for the rapid detection of other targeted sites in gDNA in a homogeneous system.

Construction of nano receptors for ubiquitin and ubiquitinated proteins based on the region-specific interactions between ubiquitin and polydopamine.

Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine. Taking advantage of this inherent binding property, we have constructed surface-imprinted magnetic nanoparticles (NPs) for ubiquitin by sequential dopamine polymerization and surface PEGylation. The obtained molecularly imprinted polymer (MIP) NPs showed a binding constant of 2.6 × 106 L mol-1 for the template ubiquitin. The bound ubiquitin could be quantitatively released by heating to 70 °C at pH 2.0 or 90 °C at neutral (pH 7.0) conditions. The MIP NPs exhibited nano receptor-like property which not only effectively blocked the formation of branched ubiquitin chains but also selectively separated ubiquitin from the bacterial cell lysates. By incubating the MIP NPs with the lysates of 293T cells, totally 529 ubiquitinated proteins were captured, among which 287 proteins were not identified by the anti-ubiquitin monoclonal antibodies (mAbs). With the distinct merits of low cost and high stability, the as-prepared MIP NPs may be utilized either separately or as an important complement to the mAbs for the purification and enrichment of ubiquitin and ubiquitinated proteins from complex biological samples. Furthermore, due to the flexibility in modification of the binding sites during or after the imprinting reactions, the results of this work also paved the way for generation of artificial receptors for branched ubiquitin chains and polyubiquitinated proteins with higher avidity and specificity.

On-line monitoring of the dopamine-based molecular imprinting processes for protein templates with the assistance of a fluorescent indicator.

On-line monitoring of the dopamine (DA)-based molecular imprinting processes over Fe3O4@SiO2-NH2 nanoparticles (SiMNPs) is reported by using a real-time quantitative PCR machine. Taking advantages of the efficient fluorescence quenching capability of polydopamine (PDA) and its high binding affinity to rhodamine B (RhB), we performed molecular imprinting against different proteins with free dopamine as the functional monomer and RhB as a fluorescent indicator. Along with the template molecules, the fluorescent indicators were continuously encapsulated into the PDA layer formed on the surface of the SiMNPs, resulting in immediate quenching of the fluorescence, which can be conveniently monitored in real time. As proteins showed sequence-dependent influences on the oxidation of dopamine and subsequent self-assembly on the surface of the SiMNPs, the observed fluorescence signals clearly indicated the polymerization progress in the presence of the template proteins, allowing precise control of the reaction time for different templates at a given initial concentration. The optimum end point of the reaction was found to be when 90 ± 3% of the templates had been encapsulated into the polymer, which offered the highest imprinting factor and selectivity. We applied the approach to prepare a primary PDA-based surface imprinted polymer for a multifunctional protein apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). After further introduction of 3-hydroxyphenylboronic acid to the interfaces between APE1 and PDA, the resultant molecularly imprinted polymers (MIP-II) enabled quantitative isolation APE1 from cell lysate samples. The developed approach will be useful for the quantitative preparation of PDA-based MIPs for precious template proteins with limited input quantity. It is also applicable for further study on the effects of different proteins or peptides on the PDA formation reactions.

Discovery of c.577del in EPO: Investigations into endogenous EPO double-band detected in blood with SAR-PAGE.

Recently, some athletes were repetitively found to have rEPO positive results, including a characterized double-band pattern in blood samples, in routine doping analysis. In contrast to previous findings from excretion studies, this double-band pattern showed the same relative intensity even when the samples were collected weeks (/months) apart. We therefore suspected that these "positive" doping control samples were related with a novel pathway of endogenous EPO production. Thus, follow-up investigations were warranted to characterize the origin of such analytical test results and to avoid the issuing of adverse analytical findings in the absence of rEPO by identifying the root cause of these "constantly positives." In this study, we designed and conducted a series of causal studies, including population screening of EPO profiles, exploration of EPO de-N-glycosylation, single nucleotide polymorphism (SNP) browsing in EPO, sequencing of EPO exons, genealogical analysis of the c.577del EPO variant, and finally expression and investigation of mutant EPO. In summary, we found that these "constantly positives" were related to endogenous EPO production associated with the c.577del EPO variant. The frequency of this variant was 0.39% in our Chinese population pool. The mutant EPO encoded by this variant is 27 amino acids longer than the wild-type. The molecular weight of this mutant EPO is approximately the same as that of rEPO, exhibiting a similar electrophoretic behavior. To prevent charges against carriers of the c.577del variant, a revised rEPO testing strategy has been implemented in the new version of TD EPO.

Circulating trophoblast cell clusters for early detection of placenta accreta spectrum disorders.

Placenta accreta spectrum (PAS) is a high-risk obstetrical condition associated with significant morbidity and mortality. Current clinical screening modalities for PAS are not always conclusive. Here, we report a nanostructure-embedded microchip that efficiently enriches both single and clustered circulating trophoblasts (cTBs) from maternal blood for detecting PAS. We discover a uniquely high prevalence of cTB-clusters in PAS and subsequently optimize the device to preserve the intactness of these clusters. Our feasibility study on the enumeration of cTBs and cTB-clusters from 168 pregnant women demonstrates excellent diagnostic performance for distinguishing PAS from non-PAS. A logistic regression model is constructed using a training cohort and then cross-validated and tested using an independent cohort. The combined cTB assay achieves an Area Under ROC Curve of 0.942 (throughout gestation) and 0.924 (early gestation) for distinguishing PAS from non-PAS. Our assay holds the potential to improve current diagnostic modalities for the early detection of PAS.

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis.

Fluorescence-based single-molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labeling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labeling sites, experimental setup, and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

Programmable One-Pot Enzymatic Reaction for Direct Fluorescence Detection of Ultralow-Abundance Mutations in the DNA Duplex.

Sensitive detection of low-abundance driver mutations may provide valuable information for precise clinical treatment. Compared to next-generation sequencing and droplet digital PCR methods, fluorescent probes show great flexibility in rapid detection of specific mutations with high sensitivity and easily accessible instruments. However, existing approaches with fluorescent probes need an additional step to convert duplex DNA to single-stranded DNA (ssDNA) before the detection step, which increases the time, cost, and risk of loss of low-input target strands. In this work, we attempt to integrate the ssDNA-generation step with the subsequent detection into a programable one-pot reaction by employing lambda exonuclease (λ exo), a versatile nanopore nuclease which exercises different functions on different substrates. The capability of λ exo in discrimination of mismatched bases in 5'- FAM-ended 2 nt-unpaired DNA duplexes was first demonstrated. Specific fluorescent probes were developed for EGFR exon 19 E746-A750del and PIK3CA E545K mutations with discrimination factors as high as 8470 and 884, respectively. By mixing the probes and λ exo with the PCR products of cell-free circulating DNA extracted from plasma samples, the reaction was immediately initiated, which allowed sensitive detection of the two types of mutations at an abundance as low as 0.01% within less than 2 h. Compared to existing approaches, the new method has distinct advantages in simplicity, low cost, and rapidity. It provides a convenient tool for companion diagnostic tests and other routine analysis targeting genetic mutations in clinical samples.

Major ascaroside pheromone component asc-C5 influences reproductive plasticity among isolates of the invasive species pinewood nematode.

Pheromones are communication chemicals and regulatory signals used by animals and represent unique tools for organisms to mediate behaviors and make "decisions" to maximize their fitness. Phenotypic plasticity refers to the innate capacity of a species to tolerate a greater breadth of environmental conditions across which it adapts to improve its survival, reproduction, and fitness. The pinewood nematode, Bursaphelenchus xylophilus, an invasive nematode species, was accidentally introduced from North America into Japan, China, and Europe; however, few studies have investigated its pheromones and phenotypic plasticity as a natural model. Here, we demonstrated a novel phenomenon, in which nematodes under the condition of pheromone presence triggered increased reproduction in invasive strains (JP1, JP2, CN1, CN2, EU1, and EU2), while it simultaneously decreased reproduction in native strains (US1 and US2). The bidirectional effect on fecundity, mediated by presence/absence of pheromones, is henceforth termed pheromone-regulative reproductive plasticity (PRRP). We further found that synthetic ascaroside asc-C5 (ascr#9), the major pheromone component, plays a leading role in PRRP and identified 2 candidate receptor genes, Bxydaf-38 and Bxysrd-10, involved in perceiving asc-C5. These results suggest that plasticity of reproductive responses to pheromones in pinewood nematode may increase its fitness in novel environments following introduction. This opens up a new perspective for invasion biology and presents a novel strategy of invasion, suggesting that pheromones, in addition to their traditional roles in chemical signaling, can influence the reproductive phenotype among native and invasive isolates. In addition, this novel mechanism could broadly explain, through comparative studies of native and invasive populations of animals, a potential underlying factor behind of the success of other biological invasions.

Fluorescence imaging of intracellular nucleases-A review.

Nucleases play crucial roles in maintaining genomic integrity. Visualization of intracellular distribution and translocation of nucleases are of great importance for understanding the in-vivo physiological functions of these enzymes and their roles in DNA repair and other cellular signaling pathways. Here we review the recently developed approaches for fluorescence imaging of nucleases in various eukaryotic cells. We mainly focused on the immunofluorescence techniques, the genetically encoded fluorescent probes and the chemically synthesized fluorescent DNA-substrate probes that enabled in-situ visualization of the subcellular localization of nucleases and their interactions with other protein/DNA molecules within cells. The targeted nucleases included important endonucleases, 3' exonucleases and 5' exonucleases that were involved in the DNA damage repair pathways and the intracellular DNA degradation. The advantages and limitations of the available tools were summarized and discussed.

Conformational Dynamics of the Periplasmic Chaperone SurA.

The periplasmic protein SurA is the primary chaperone involved in the biogenesis of bacterial outer membrane proteins and is a potential antibacterial drug target. The three-dimensional structure of SurA can be divided into three parts, a core module formed by the N- and C-terminal regions and two peptidyl-prolyl isomerase (PPIase) domains, P1 and P2. Despite the determination of the structures of several SurA-peptide complexes, the functional mechanism of this chaperone remains elusive and the roles of the two PPIase domains are yet unclear. Herein, we characterize the conformational dynamics of SurA by using solution nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer methods. We demonstrate a "closed-to-open" structural transition of the P1 domain that is correlated with both chaperone activity and peptide binding and show that the flexible P2 domain can also occupy conformations that closely contact the NC core module. Our results offer a structural basis for the counteracting roles of the two PPIase domains in regulating the SurA chaperone activity.

Construction of Specific and Reversible Nanoreceptors for Proteins via Sequential Surface-Imprinting Strategy.

Molecular recognition of proteins is critical for study and manipulation of protein-related biological processes. However, design and synthesis of abiotic receptors for precise recognition of proteins still remains a challenging task. Herein, we developed a universal sequential surface-imprinting strategy that integrated two different types of imprinting reactions to construct artificial protein receptors with high selectivity. Employing dopamine self-polymerization and boronate/diol complexation as the first-step and second-step imprinting reactions, respectively, we synthesized surface-imprinted magnetic nanocomposites against two different enzyme proteins: deoxyribonuclease I (DNase I) and apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). The obtained nanocomposites both showed strong and specific binding toward their respective template proteins. Moreover, the bound enzymes could be totally recovered with high activity under mild buffer conditions. These antibody-like specific and reversible binding properties enabled effective purification and enrichment of the low-abundance target proteins from complex serum samples. Compared to existing one-pot or one-step imprinting methods, the proposed sequential surface-imprinting approach offers a more flexible combination of different functional monomers and greatly enhances the performance and biocompatibility of the imprinted materials. The generality and simplicity of the sequential imprinting strategy would make it an appealing and competitive method to prepare artificial protein receptors.

Occurrence and Characterization of Fungi and Mycotoxins in Contaminated Medicinal Herbs.

Traditional medicinal herbs are widely used and may be contaminated with mycotoxigenic fungi during cultivation, harvesting, and storage, causing spoilage and mycotoxin production. We evaluated the predominant mycoflora and extent of mycotoxin contaminations in 48 contaminated samples of 13 different medicinal herbs. In total, 70.8% of herbs were slightly contaminated with aflatoxins (<5 µg kg-1). Codonopsis radix samples contained ochratoxin A (OTA) (360-515 µg kg-1), and Scutellariae radix samples contained OTA (49-231 µg kg-1) and citrinin (15-53 µg kg-1). Forty samples (83.3%) contained fungal contamination. Sixty-nine strains were characterized via morphological and molecular identification. The predominant mycoflora comprised four genera, Aspergillus spp. (26.1%), Penicillium spp. (24.6%), Rhizopus spp. (14.5%), and Trichoderma spp. (11.6%). Aflatoxins, OTA, and citrinin were detected in 37 cultures by high-performance liquid chromatography-tandem mass spectrometry. Approximately 21.6% of Aspergillus and Penicillium isolates produced mycotoxins. One Penicillium polonicum strain isolated from Scutellariae radix synthesized citrinin. Multiplex PCR analysis showed that three Aspergillus flavus strains harbored aflatoxin biosynthesis genes. One Aspergillus flavus strain isolated from Amomi fructus produced AFB1 and AFB2. To the best of our knowledge, the citrinin production by Aspergillus chevalieri and Penicillium sacculum was first reported in this study, which poses a potential risk of mycotoxin contamination in medicinal herbs.