Dynamics of the content of reactive oxygen species and the state of the glutathione system in the oral cavity during subchronic intoxication wuth the fungicide thiram and its antioxidant correction.

Thiram is a dithiocarbamate derivative, which is used as a fungicide for seed dressing and spraying during the vegetation period of plants, and also as an active vulcanization accelerator in the production of rubber-based rubber products. In this study the content of reactive oxygen species (ROS) and the state of the glutathione system have been investigated in the oral fluid and gum tissues of adult male Wistar rats treated with thiram for 28 days during its administration with food at a dose of 1/50 LD50. Thiram induced formation of ROS in the oral cavity; this was accompanied by an imbalance in the ratio of reduced and oxidized forms of glutathione due to a decrease in glutathione and an increase in its oxidized form as compared to the control. Thiram administration caused an increase in the activity of glutathione-dependent enzymes (glutathione peroxidase, glutathione transferase, and glutathione reductase). However, the time-course of enzyme activation in the gum tissues and oral fluid varied in dependence on the time of exposure to thiram. In the oral fluid of thiram-treated rats changes in the antioxidant glutathione system appeared earlier. The standard diet did not allow the glutathione pool to be fully restored to physiological levels after cessation of thiram intake. The use of exogenous antioxidants resviratrol and an Echinacea purpurea extract led to the restoration of redox homeostasis in the oral cavity.

Molecular mechanism of miR-203a targeting Runx2 to regulate thiram induced-chondrocyte development.

Thiram is a kind of organic compound, which is commonly used for sterilization, insecticidal and deodorization in daily life. Its toxicology has been broadly studied. Recently, more and more microRNAs have been shown to participate in the regulation of cartilage development. However, the potential mechanism by which microRNA regulates chondrocyte growth is still unclear. Our experiments have demonstrated that thiram can hamper chondrocytes development and cause a significant increase in miR-203a content in vitro and in vivo trials. miR-203a mimic significantly decrease in mRNA and protein expression of Wnt4, Runx2, COL2A1, ß-catenin and ALP, and significantly enhance the mRNA and protein levels of GSK-3ß. It has been observed that overexpression of miR-203a hindered chondrocytes development. In addition, Runx2 was confirmed to be a direct target of miR-203a by dual luciferase report gene assay. Transfection of si-Runx2 into chondrocytes reveals that significant downregulation of genes is associated with cartilage development. Overall, these results suggest that overexpression of miR-203a inhibits the expression of Runx2. These findings are conducive to elucidate the mechanism of chondrocytes dysplasia induced by thiram and provide new research ideas for the toxicology of thiram.

Leucine improves thiram-induced tibial dyschondroplasia and gut microbiota dysbiosis in broilers.

Thiram, a commonly used agricultural insecticide and fungicide, has been found to cause tibial dyschondroplasia (TD) in broilers, leading to substantial economic losses in the poultry industry. In this study, we aimed to investigate the mechanism of action of leucine in mitigating thiram-induced TD and leucine effects on gut microbial diversity. Broiler chickens were randomly divided into five equal groups: control group (standard diet), thiram-induced group (thiram 80 mg/kg from day 3 to day 7), and different concentrations of leucine groups (0.3%, 0.6%, 0.9% leucine from day 8 to day 18). Performance indicator analysis and tibial parameter analysis showed that leucine positively affected thiram-induced TD broilers. Additionally, mRNA expressions and protein levels of HIF-1α/VEGFA and Ihh/PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. The results showed that leucine recovered lameness disorder by downregulating the expression of HIF-1α, VEGFA, and PTHrP while upregulating the expression of Ihh. Moreover, the 16 S rRNA sequencing revealed that the leucine group demonstrated a decrease in the abundance of harmful bacteria compared to the TD group, with an enrichment of beneficial bacteria responsible for producing short-chain fatty acids, including Alistipes, Paludicola, CHKCI002, Lactobacillus, and Erysipelatoclostridium. In summary, the current study suggests that leucine could improve the symptoms of thiram-induced TD and maintain gut microbiota homeostasis.

Flexible, scalable and simple-fabricated silver nanorod-decorated bacterial nanocellulose SERS substrates cooperated with portable Raman spectrometer for on-site detection of pesticide residues.

Owing to good flexibility, prominent mechanical properties, three-dimensional (3D) nanofibrous structure and low background interference, sustainable bacterial nanocellulose (BNC) is a highly attractive matrix material for surface-enhanced Raman scattering (SERS) sensor. Herein, a highly sensitive, flexible and scalable silver nanorod-decorated BNC (AgNRs@BNC) SERS sensor is developed by a simple vacuum-assisted filtration. The AgNRs were firmly locked in the 3D nanofibrous network of cellulose nanofibers upon vacuum drying process, resulting in the formation of 3D SERS hotspots with a depth of more than 10 µm on the sensor. With 4-aminothiophenol (4-ATP) as a target molecule, a lowest distinguishable level of 10-12 M and a high enhancement factor of 1.1 × 109 were realized by the optimal AgNRs1.5@BNC SERS sensor. Moreover, the AgNRs@BNC SERS sensor exhibits high detectable level of 10-9 M for thiram molecules by integrating with a portable Raman spectrometer. Besides, toxic thiram residues on grape surface could be directly on-site identified by the combination of AgNRs@BNC SERS sensors and a portable Raman spectrometer through a feasible press-and-peel method. The flexible AgNRs@BNC SERS sensor cooperated with portable Raman system demonstrates great potential for on-site detection of pesticide residues on irregular food surfaces.

Molecular mechanism of thiram-induced abnormal chondrocyte proliferation via lncRNA MSTRG.74.1-BNIP3 axis.

Thiram, a widely used organic pesticide in agriculture, exhibits both bactericidal and insecticidal effects. However, prolonged exposure to thiram has been linked to bone deformities and cartilage damage, contributing to the development of tibial dyschondroplasia (TD) in broilers and posing a significant threat to global agricultural production. TD, a prevalent nutritional metabolic disease, manifests as clinical symptoms like unstable standing, claudication, and sluggish movement in affected broilers. In recent years, there has been growing recognition of the regulatory role of long non-coding RNA (lncRNA) in tibial cartilage formation among broilers through diverse signaling pathways. This study employs in vitro experimental models, growth performance analysis, and clinical observation to assess broilers' susceptibility to thiram pollution. Transcriptome sequencing analysis revealed a significant elevation in the expression of lncRNA MSTRG.74.1 in both the con group and the thiram-induced in vitro group. The results showed that lncRNA MSTRG.74.1 plays a pivotal role in influencing the proliferation and abnormal differentiation of chondrocytes. This regulation occurs through the negative modulation of apoptotic genes, including Bax, Cytc, Bcl2, Apaf1, and Caspase3, along with genes Atg5, Beclin1, LC3b, and protein p62. Moreover, the overexpression of lncRNA MSTRG.74.1 was found to regulate broiler chondrocyte development by upregulating BNIP3. In summary, this research sheds light on thiram-induced abnormal chondrocyte proliferation in TD broilers, emphasizing the significant regulatory role of the lncRNA MSTRG.74.1-BNIP3 axis, which will contribute to our understanding of the molecular mechanisms underlying TD development in broilers exposed to thiram.

The autophagy-mediated mechanism via TSC1/mTOR signaling pathway in thiram-induced tibial dyschondroplasia of broilers.

Thiram is a member of the dithiocarbamate family and is widely used in agriculture, especially in low-income countries. Its residues lead to various diseases, among which tibial dyschondroplasia (TD) in broiler chickens is the most common. Recent studies have also demonstrated that thiram residues may harm human health. Our previous study showed that the activity of the mTOR (mammalian target of rapamycin) signaling pathway has changed after thiram exposure. In the current study, we investigated the effect of autophagy via the mTOR signaling pathway after thiram exposure in vitro and in vivo. Our results showed that thiram inhibited the protein expression of mTOR signaling pathway-related genes such as p-4EBP1 and p-S6K1. The analysis showed a significant increase in the expression of key autophagy-related proteins, including LC3, ULK1, ATG5, and Beclin1. Further investigation proved that the effects of thiram were mediated through the downregulation of mTOR. The mTOR agonist MHY-1485 reverse the upregulation of autophagy caused by thiram in vitro. Moreover, our experiment using knockdown of TSC1 resulted in chondrocytes expressing lower levels of autophagy. In conclusion, our results demonstrate that thiram promotes autophagy via the mTOR signaling pathway in chondrogenesis, providing a potential pharmacological target for the prevention of TD.

Comprehensive Identification and Ubiquitous Occurrence of Eight Classes of Rubber-Derived Vulcanization Accelerators in Urban Dusts.

Vulcanization accelerators (VAs) serve as crucial additives in synthetic rubber on a global scale. Despite their widespread use, the environmental presence, distribution, and associated exposure risks of VAs remain poorly understood. This study compiled a target list and conducted a screening for eight classes encompassing 42 VAs in diverse urban dust samples from South China. A total of 40 of the 42 target VAs were detectable across all four studied regions, among which 30 were identified for the first time in the environment. Among the eight structure-classified VA classes, xanthates exhibited the highest concentrations (median: 3810-81,300 ng/g), followed by thiazoles, guanidines, sulfenamides, dithiocarbamates, thiurams, thioureas, and others. The median total concentrations of all target VAs (∑VAs) were determined to be 5060 ng/g in road dust, 5730 ng/g in parking lot dust, 29,200 ng/g in vehicle repair plant dust, and 84,300 ng/g in household dust, indicating the widespread presence of numerous rubber-derived VAs in various urban environments. This study marked the first systematic effort to identify a wide range of emerging rubber-derived VAs prevalent in urban environments. The findings call for increased attention to these widely utilized but less well-evaluated chemicals in future research and environmental management efforts.

Ag-modified CuO cavity arrays as a SERS-electrochemical dual signal platform for thiram detection.

Rapid and sensitive determination of pesticide residues in fruits and vegetables is critical for human health and ecosystems. This paper used an Ag-modified CuO sphere-cavity array (CuO@Ag) electrode as a thiram SERS/electrochemical dual readout detection platform. Numerous Raman "hotspots" generated by uniformly distributed silver nanoparticles, charge transfer at the CuO@Ag interface, and the formation of Ag-thiram complexes contribute to the significant enhancement of this SERS substrate, which results in excellent SERS performance with an enhancement factor up to 1.42 × 106. When using SERS as the readout technique, the linear range of the substrate for thiram detection was 0.05-20 nM with a detection limit (LOD) of up to 0.0067 nM. Meanwhile, a correlation between the value of change in current density and thiram concentration was established due to the formation of stable complexes of thiram with Cu2+ generated at specific potentials. The linear range of electrochemical detection was 0.05-20.0 µM, and the detection limit was 0.0167 µM. The newly devised dual-readout sensor offers notable sensitivity and stability. The two signal readout methods complement each other in terms of linear range and detection limit, making it a convenient tool for assessing thiram residue levels in agro-food. At the same time, the combination of commercially available portable equipment makes on-site monitoring possible.

Effects of commonly used carbamates (carbaryl and thiram) on the regulatory, secretory and motor functions of bovine cervixes in vitro.

Previously studied classes of pesticides, including organochlorines, organophosphates and pyrethroids disturb the mechanism that causes bovine myometrial contractions. Hence, the aim of this study was to investigate the effects of carbaryl and thiram, which are representative carbamate pesticides commonly used in global agriculture, on the motor and secretory functions of bovine cervixes. Additionally, the impacts of these pesticides on intra- and intercellular signaling in vitro were estimated. In this study, cervical cells or strips were obtained from cows at days 18-20 of the estrous cycle and were treated with carbaryl or thiram. Neither carbamate (10 or 100 ng/ml) exerted cytotoxic effects. Carbaryl increased the level of mRNA (at a dose of 0.1 ng/ml) and protein (at both doses, 1 and 10 ng/ml) expression for the oxytocin receptor (OXTR), while thiram (at 0.1 and 10 ng/ml or 0.1-10 ng/ml, respectively) caused the opposite effects. Moreover, the level of the second messenger inositol-trisphosphate (IP3) was decreased by carbaryl (10 ng/ml) but increased by thiram (10 ng/ml). Only thiram decreased prostaglandin-endoperoxide synthase 2 (PTGS2; 0.1 ng/ml) and aldo-keto reductase family 1, member B1 (AKR1B1; 0.1 ng/ml), and prostaglandin E synthase 2 (PTGES2; 0.1-10 ng/ml) mRNA expression, while thiram (0.1-10 ng/ml) and carbaryl (0.1 and 10 ng/ml) both decreased the release of PGF2α. Carbaryl (10 ng/ml) and thiram (10 ng/ml) also decreased the level of a gap junction protein (GAP). Moreover, carbaryl (10 ng/ml) decreased the level of myosin light chain kinase (MLCK). However, the strength of cervical contractions was increased by thiram (1 and 10 ng/ml) but decreased by carbaryl (1 and 10 ng/ml). Carbaryl increased the receptivity of cervical cells to oxytocin (OXT), but inhibited further transduction (IP3) of this signal. Hence, direct inhibition of cervical strip contraction may occur. In contrast, thiram mostly decreased the receptivity of cervical cells to OXT, while it stimulated the contraction of cervical strips. Moreover, compared to carbaryl, thiram more greatly affected the synthesis and release of prostaglandins. These results suggest that carbaryl and thiram disturb OXT signaling, PG secretion and cervical contraction in vitro.

Preparation of cellulose-based flexible SERS and its application for rapid and ultra-sensitive detection of thiram on fruits and vegetables.

In response to the prevalent issue of thiram as a common pesticide residue on the surface of fruits and vegetables, our research team employed an acidic hydrated metal salt low co-fusion solvent to dissolve cellulose lysis slurry. Subsequently, a regenerated cellulose membrane (RCM) was successfully prepared via sol-gel method. Uniformly sized Ag nanoparticles (NPs) were deposited on RCM utilizing the continuous ion layer adsorption and reaction (SILAR) technique. The resulting Ag NPs/RCM flexible surface-enhanced Raman spectroscopy (SERS) substrates exhibited a minimum detection limit of 5 × 10-9 M for Rhodamine 6G (R6G), demonstrating good uniformity (RSD = 4.86 %) and reproducibility (RSD = 3.07 %). Moreover, the substrate displayed a remarkable sensitivity of 10-10 M toward thiram standard solution. Given its inherent flexibility, the substrate proves advantageous for the detection of three-dimensional environments such as fruit and vegetable surfaces, and its practicality has been confirmed in the detection of thiram residue on apples, tomatoes, pears, and other fruits and vegetables.

G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Green and sustainable self-cleaning flexible SERS base: Utilized for cyclic-detection of residues on apple surface.

Advances in flexible SERS substrates has made it possible to approach the ultimate goal of rapid in-situ monitoring of fruit and vegetable safety, but its vulnerability under laser ablation results in low utilization. In order to solve this problem, a 3D framework of TiO2-doped PVDF\PVP polymer was utilized to self-assemble gold-silver core-shell nanorods (Au@Ag NRs) to prepare a flexible SERS substrate with good physical stability and self-cleaning properties. This substrate showed excellent detection limit and recyclability after the detection of three pesticide residues in apple peel. The LOD of methyl-parathion (MP) was as low as 0.037 ng/cm2, with an RSD of 5.61 % for 5 cycle-detection. The recoveries of two additional pesticides thiram (TMTD) and chlorpyrifos (CPF) were 86.32 %-112.47 %. We hoped that this research will contribute to providing a recyclable and facile method for in-situ analysis of fruit and vegetable surface residues and functional manufacture of flexible SERS substrates.

Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

Utility of targeted mean total deviation trend analysis for detecting progressive visual field changes in early-to-moderate stage glaucoma.

OBJECTIVES: To evaluate the clinical utility of trend-based analysis of the targeted mean total deviation (TMTD) by comparing its rates of visual field (VF) change and sensitivities of detecting VF progression with those of the mean total deviation (mTD) in the global and hemifield VF area in early to-moderate glaucoma patients. METHODS: A single eye from 139 open-angle glaucoma patients with hemifield VF defects and a minimum two year follow-up were retrospectively evaluated. The TMTD was estimated by averaging the total deviation (TD) values after excluding VF points that had a threshold sensitivity of <0 dB in three baseline tests, and the mTD by averaging the entire VF TD values. The study patients were classified as VF progressors vs. non-progressors using both event- and trend-based analysis. The rates of change and ratios of progression detection were compared between TMTD and mTD. RESULTS: This study included 49 VF progressors and 90 non-progressors. Slopes for the global and VF-affected hemifield TMTD were significantly faster than those for the mTD in each subgroup and in the entire cohort (P < 0.001). Trend-based TMTD analysis detected VF progression in greater proportion than either trend-based mTD or event-based analysis (38.1% vs. 30.2% vs. 27.3%, respectively: VF affected hemifields). CONCLUSIONS: The rates of change in the TMTD are significantly faster than those for the mTD globally and in the VF-affected hemifields. Trend-based TMTD analysis shows greater sensitivity for detecting VF progression than trend-based mTD or event-based analysis in early-to-moderate glaucoma patients with hemifield VF loss.

Thiram exposure induces tibial dyschondroplasia in broilers via the regulation effect of circ_003084/miR-130c-5p/BMPR1A crosstalk on chondrocyte proliferation and differentiation.

Thiram, an agricultural insecticide, has been demonstrated to induce tibial dyschondroplasia (TD) in avian species. Circular RNA (circRNAs), a novel class of functional biological macromolecules characterized by their distinct circular structure, play crucial roles in various biological processes and diseases. Nevertheless, the precise regulatory mechanism underlying non-coding RNA involvement in thiram-induced broiler tibial chondrodysplasia remains elusive. In this study, we established a broiler model of thiram exposure for 10 days to assess TD and obtain a ceRNA network by RNA sequencing. By analyzing the differentially expressed circRNAs network, we id entify that circ_003084 was significantly upregulated in TD cartilage. Elevated circ_003084 inhibited TD chondrocytes proliferation and differentiation in vitro but promote apoptosis. Mechanistically, circ_003084 competitively binds to miR-130c-5p and prevents miR-130c-5p to decrease the level of BMPR1A, which upregulates the expression of apoptosis genes Caspase 3, Caspase 9, Bax and Bcl2, and finally facilitates cell apoptosis. Taken together, these findings imply that circ_003084/miR-130c-5p/BMPR1A interaction regulated TD chicken chondrocyte proliferation, apoptosis, and differentiation. This is the first work to reveal the mechanism of regulation of circRNA-related ceRNA on thiram-induced TD, offering a key reference for environmental toxicology.

Determination of thiram in fruit juices using a bacterial cellulose nanocrystal-based SERS substrate.

Pesticide residues in agri-foods have risk to human health and one solution is to develop simple and accurate methods for rapid detection. We developed a SERS sensor composed of gold nanoparticles (AuNPs) and bacterial cellulose nanocrystal (BCNC) to detect thiram in fruit juice. BCNC-SO3H was used as a stabilizer to support AuNPs via electrostatic repulsion, fabricating a BCNC-AuNPs SERS substrate with uniformly distributed AuNPs. This BCNC-AuNPs SERS substrate was applied to determine thiram residues in peach juice, apple juice, and grape juice with the limits of detection of 0.036 ppm, 0.044 ppm, and 0.044 ppm, respectively. The whole test took 12 min including sample preparation and analysis. The detection limits meet the maximum residue levels of thiram in fruit juices required by China, Europe and North America, indicating that this BCNC-based substrate could serve as a satisfactory SERS sensor for pesticide residue monitoring in the food supply chain.

Kinetic and structural details of urease inactivation by thiuram disulphides.

This paper reports on the molecular details of the reactivity of urease, a nickel-dependent enzyme that catalyses the last step of organic nitrogen mineralization, with thiuram disulphides, a class of molecules known to inactivate the enzyme with high efficacy but for which the mechanism of action had not been yet established. IC50 values of tetramethylthiuram disulphide (TMTD or Thiram) and tetraethylthiuram disulphide (TETD or Disulfiram) in the low micromolar range were determined for plant and bacterial ureases. The X-ray crystal structure of Sporosarcina pasteurii urease inactivated by Thiram, determined at 1.68 Å resolution, revealed the presence of a covalent modification of the catalytically essential cysteine residue. This is located on the flexible flap that modulates the size of the active site channel and cavity. Formation of a Cys-S-S-C(S)-N(CH3)2 functionality responsible for enzyme inactivation was observed. Quantum-mechanical calculations carried out to rationalise the large reactivity of the active site cysteine support the view that a conserved histidine residue, adjacent to the cysteine in the active site flap, modulates the charge and electron density along the thiol SH bond by shifting electrons towards the sulphur atom and rendering the thiol proton more reactive. We speculate that this proton could be transferred to the nickel-coordinated urea amide group to yield a molecule of ammonia from the generated Curea-NH3+ functionality during catalysis.

Analyte-induced laccase-mimicking activity inhibition and conductivity enhancement of electroactive nanozymes for ratiometric electrochemical detection of thiram.

Most nanozyme-based electrochemical sensing strategies depend on the catalytic formation of electroactive substances, while the electrochemical properties of nanozymes have rarely been explored. In this study, magnetic nanoparticles encapsulated metal-organic framework served as precursors to prepare bioinspired nanozymes with both laccase-mimicking activity and electroactivity. Owing to the strong affinity between thiram (THR) and Cu(II) active sites in the nanozymes, the binding of THR inhibited nanozyme catalytic activity toward catechol (CT) oxidation and enhanced nanozyme conductivity. A lower oxidation current (ICT) of CT was accompanied by a higher oxidation signal (ICu) of Cu(II), allowing a ratiometric electrochemical response of the electroactive nanozymes toward the incoming THR. The signal ratio (ICu/ICT) displayed a good linear relationship over a THR concentration range of 10.0 nM-3.0 µM with a limit of detection of 0.15 nM, and the entire THR detection process was rapidly accomplished within 5 min. The high sensitivity and selectivity of the developed electrochemical strategy guaranteed the reliable detection of THR in fruit, vegetable, and river water samples. This study provides new insights into the development of nanozymes for electrochemical analysis.

Low-dose thiram exposure elicits dysregulation of the gut microbial ecology in broiler chickens.

Thiram, a typical fungicide pesticide, is widely used in agricultural production. The presence of thiram residues is not only due to over-utilization, but is also primarily attributed to long-term accumulation. However, there is a paucity of information regarding the impact of prolonged utilization of thiram at low doses on the gut microbiota, particularly with respect to gut fungi. Our objective is to explore the effect of thiram on broilers from the perspective of gut microbiota, which includes both bacteria and fungi. We developed a long-term low-dose thiram model to simulate thiram residue and employed 16 S rRNA and ITS gene sequencing to investigate the diversity and profile of gut microbiota between group CC (normal diet) and TC (normal diet supplemented with 5 mg/kg thiram). The results revealed that low doses of thiram had a detrimental effect on broiler's growth performance, resulting in an approximate reduction of 669.33 g in their final body weight at day 45. Our findings indicated that low-dose thiram had a negative impact on the gut bacterial composition, leading to a notable reduction in the abundance of Merdibacter, Paenibacillus, Macrococcus, Fournierella, and Anaeroplasma (p < 0.05) compared to the CC group. Conversely, the relative level of Myroides was significantly increased (p < 0.05) in response to thiram exposure. In gut fungi, thiram significantly enhanced the diversity and richness of gut fungal populations (p < 0.05), as evidenced by the notable increase in alpha indices, i.e. ACE (CC: 346.49 ± 117.27 vs TC: 787.27 ± 379.14, p < 0.05), Chao 1 (CC: 317.63 ± 69.13 vs TC: 504.85 ± 104.50, p < 0.05), Shannon (CC: 1.28 ± 1.19 vs TC: 5.39 ± 2.66, p < 0.05), Simpson (CC: 0.21 ± 0.21 vs TC: 0.78 ± 0.34, p < 0.05). Furthermore, the abundance of Ascomycota, Kickxellomycota, and Glomeromycota were significantly increased (p < 0.05) by exposure to thiram, conversely, the level of Basidiomycota was decreased (p < 0.05) in the TC group compared to the CC group. Overall, this study demonstrated that low doses of thiram induced significant changes in the composition and abundance of gut microbiota in broilers, with more pronounced changes observed in the gut fungal community as compared to the gut bacterial community. Importantly, our findings further emphasize the potential risks associated with low dose thiram exposure and have revealed a novel discovery indicating that significant alterations in gut fungi may serve as the crucial factor contributing to the detrimental effects exerted by thiram residues.

miR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/ß-catenin pathway.

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/ß-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/ß-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, ß-catenin and Col2a1 increased but the expression of GSK-3ß decreased. It was observed that inhibition of WIF1 increased the expression of ALP, ß-catenin, Col2a1 and ACAN but decreased the expression of GSK-3ß. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/ß-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.