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An electrochemical biosensor based on a polystyrene-gold nanocomposite was fabricated for the ultrasensitive sensing of clenbuterol. The PS-Au nanocomposite was synthesized by modifying polystyrene with triazine and aminothiazole groups, followed by the in-situ reduction of AuNPs. The PS-Au/SPCE exhibited a linear range (0.01–10 μM) with a limit of detection of 3.2 nM towards clenbuterol along with high selectivity and good stability. The sensor platform showcased its prowess by accurately quantifying clenbuterol in milk specimens fortified with known analyte concentrations, yielding recovery percentages within the range of 96.5–102.8 %, accompanied by relative standard deviations below 4.2 %. The superior performance of the PS-Au/SPCE is due to the synergistic effect of the high surface area and electrocatalytic activity of the AuNPs, the efficient adsorption of clenbuterol on the functionalized polystyrene matrix, and the enhanced electron transfer kinetics. [ABSTRACT FROM AUTHOR]

Aims: Cerebral hypometabolism occurs years prior to a diagnosis of neurodegenerative diseases and coincides with reduced cerebral perfusion and declining noradrenergic transmission from the locus coeruleus. In pre‐clinical models, β‐adrenoceptor (β‐AR) agonists increase cerebrocortical glucose metabolism, and may have therapeutic potential for neurodegenerative diseases. This study investigated the safety and effects on regional cerebral blood flow (rCBF) of the oral, brain‐penetrant β2‐AR agonist, clenbuterol, in healthy volunteers (HV) and patients with mild cognitive impairment (MCI) or Parkinson's disease (PD). Methods: This study evaluated the safety and effects on cerebral activity of the oral, brain‐penetrant, β2‐AR agonist clenbuterol (20–160 μg) in healthy volunteers and patients with MCI or PD. Regional CBF, which is tightly coupled to glucose metabolism, was measured by arterial spin labelling MRI in 32 subjects (25 HV and 8 MCI or PD) across five cohorts. In some cohorts, low doses of nadolol (1–5 mg), a β‐AR antagonist with minimal brain penetration, were administered with clenbuterol to control peripheral β2‐AR responses. Results: Significant, dose‐dependent increases in rCBF were seen in multiple brain regions, including hippocampus, amygdala and thalamus, following the administration of clenbuterol to HVs (mean changes from baseline in hippocampal rCBF of −1.7%, 7.3%, 22.9%, 28.4% 3 h after 20, 40, 80 and 160 μg clenbuterol, respectively). In patients with MCI or PD, increases in rCBF following 80 μg clenbuterol were observed both without and with 5 mg nadolol (in hippocampus, 18.6%/13.7% without/with nadolol). Clenbuterol was safe and well‐tolerated in all subjects; known side effects of β2‐agonists, including increased heart rate and tremor, were mild in intensity and were blocked by low‐dose nadolol. Conclusions: The effects of clenbuterol on rCBF were evident both in the absence and presence of low‐dose nadolol, suggesting central nervous system (CNS) involvement. Concomitant inhibition of the peripheral effects of clenbuterol by nadolol confirms that meaningful β2‐AR antagonism in the periphery was achieved without interrupting the central effects of clenbuterol on rCBF. [ABSTRACT FROM AUTHOR]

Background: Noradrenergic signaling declines in Parkinson's disease (PD) following locus coeruleus neurodegeneration. Epidemiologic studies demonstrate that β-acting drugs slow PD progression. Objective: The primary objective was to compare the safety and effects of 3 β-adrenoceptor (β-AR) acting drugs on central nervous system (CNS) function after a single dose in healthy volunteers (HVs) and evaluate the effects of multiple doses of β-AR acting drugs in HVs and PD-patients. Methods: In Part A, HVs received single doses of 32 mg salbutamol, 160μg clenbuterol, 60 mg pindolol and placebo administered in a randomized, 4-way cross-over study. In Part B (randomized cross-over) and Part C (parallel, 2:1 randomized), placebo and/or clenbuterol (20μg on Day 1, 40μg on Day 2, 80μg on Days 3–7) were administered. CNS functions were assessed using the NeuroCart test battery, including pupillometry, adaptive tracking and recall tests. Results: Twenty-seven HVs and 12 PD-patients completed the study. Clenbuterol improved and pindolol reduced the adaptive tracking and immediate verbal recall performance. Clenbuterol and salbutamol increased and pindolol decreased pupil-to-iris ratios. Clenbuterol was selected for Parts B and C. In Part B, clenbuterol significantly increased performance in adaptive tracking with a tendency toward improved performance in immediate and delayed verbal recall. In Part C trends toward improved performance in immediate and delayed verbal recall were observed in PD-patients. Typical cardiovascular peripheral β2-AR effects were observed with clenbuterol. Conclusions: This study demonstrates the pro-cognitive effects of clenbuterol in HVs with similar trends in PD-patients. The mechanism of action is likely activation of β2-ARs in the CNS. Plain Language Summary: Aims and Purpose of the Research: This research aimed to explore how three different drugs affect brain function. These drugs are salbutamol, clenbuterol, and pindolol and work in the brain by stimulating specific brain cells that can improve aspects like memory and coordination. The main question was to see how safe these drugs were and how they impact the brain function after one dose in healthy people, and after multiple doses in both healthy people and those with Parkinson's disease. Background of the Research: Parkinson's disease is a condition where brain cells start to die, which affects different areas of the brain, including movement function, as well as memory and attention. This research matters because finding drugs that affect the brain function could improve the lives of people with Parkinson's disease. Methods and Research Design: The study was conducted in three parts. In the first part, healthy volunteers took one dose of each of the three drugs— salbutamol, clenbuterol, and pindolol— as well as a placebo (a harmless pill that has no effect). The researchers tested the participants' brain functions using various tasks including memory tests and eye response measurements. In the second and third part, healthy people and people with Parkinson's disease took the drug that performed best in healthy volunteers for seven days. Results and Importance: In the first part, a single dose of clenbuterol was safe and improved memory and attentions tasks in healthy people, and therefore was chosen for further testing in the second and third part. In these parts, multiple doses of clenbuterol were safe and helped improve memory and attention tasks in healthy people, with similar positive trends seen in people with Parkinson's disease. The study suggests that clenbuterol might help improve brain function by activating specific receptors in the brain. These results are important because they suggest that clenbuterol could be a potential treatment to help improve brain function in people with Parkinson's disease. However, more research is needed to fully understand its effects and to confirm these findings. [ABSTRACT FROM AUTHOR]

In this study, a fluorescence detection method combining aptamer-modified up-conversion nanoparticles (UCNPs) and magnetic nanoparticles (MNPs) was developed for detection of Clenbuterol (CLB). The aptamer-modified magnetic NPs captured CLB, which reacted with the aptamer-modified UCNPs and generated a sandwich complex. The aptamer-modified UCNPs acted as a fluorescence source. The MNP-CLB-UCNP complex was retrieved from the solution using an magnetic field, and the fluorescence intensity was detected by fluorescence spectrophotometry with excitation and emission spectra at 980 nm and in the 400–800 nm region, respectively. The results showed that the fluorescence intensity gradually increased with increasing concentrations of CLB with a good specify. The method was highly sensitive for the quantification of CLB, with a limit of detection of 0.304 ng mL−1. The recovery rate of CLB from pork samples ranged from 84 % to 94.87 %. This fluorescence method enables the sensitive, precise, and accurate quantification of CLB residues in pork samples.

Chronic ingestion of food containing clenbuterol (CLB) in the human body can cause acute poisoning. Thus, it is essential to develop a sensitive CLB detection way. Here, a novel electrochemical aptamer sensor based on aptamer/gold nanoparticles/amino magnetic ferric oxide/graphene nanosheets (aptamer/Au NPs/Fe3O4–NH2/GN) nanocomposite has been developed. The Fe3O4–NH2 nanoparticles were self-assembled on GN, and then Fe3O4–NH2/GN nanocomposite was coated on the glassy carbon electrode (GCE) surface. Afterward, Au NPs were electrodeposited on Fe3O4–NH2/GN nanocomposite to fix the aptamer by the Au–S bonds. When CLB was added to the solution, the aptamer will specifically recognize CLB, causing current changes. Under the optimized conditions, the sensor shows better electrochemical performance (linear range is 1 × 1 0 − 1 3 M to 1 × 1 0 − 7 M, the detection limit is 1. 1 6 × 1 0 − 1 4 M (S / N = 3)). It has been successfully applied to the detection of CLB in actual samples. The results show that the aptamer sensor has good selectivity, stability and repeatability, which will provide a novel idea for more sensitive and quicker CLB detection. We developed a novel electrochemical aptamer sensor based on aptamer/Au NPs/Fe3O4–NH2/GN. The Fe3O4–NH2 nanoparticles were grown on GN, and then Fe3O4–NH2/GN nanocomposite was modified on the glassy carbon electrode (GCE) surface. Afterwards, Au NPs were electrodeposited on Fe3O4–NH2/GN to fix the aptamer by the Au–S bonds. The aptamer will change current specifically when CLB is in the detection system. [ABSTRACT FROM AUTHOR]

A highly sensitive lateral flow immunoassay (LFIA) is developed for the enzyme-catalyzed and doublereading determination of clenbuterol (CLE), in which a new type of probe was adopted through the direct electrostatic adsorption of ultra-small copper-gold bimetallic enzyme mimics (USCGs) and monoclonal antibodies. In the assay, based on the peroxidase activity of USCG, the chromogenic substrate TMB-H2O2 was introduced to trigger its color development, and the results were compared with those before catalysis. The detection sensitivity after catalysis is 0.03 ng mL-1 under optimal circumstances, which is 6-fold better than that of the traditional Au NPs-based LFIA and 2-fold greater than that before catalysis. This approach was successfully applied to the detection of CLE in milk, pork and mutton samples with an optimum assay time of 7 min and best catalytic time of 80 s, after which satisfactory recoveries of 98.53-117.79% were obtained. Cu-Au nanoparticles as a signal tag and the use of their nanozyme properties are the first applications in the field of LFIA. This work can be a promising exhibition for the application of a cheaper substitute for HRP, ultra-small bimetallic enzyme mimics, in LFIAs. [ABSTRACT FROM AUTHOR]

Background and Aim: Incorporating physical activity and a balanced diet can play a crucial role in managing and preventing obesity-related diseases. This study aimed to assess the effects of six weeks of High-Intensity Interval Training (HIIT) exercises combined with EPA/DHA and clenbuterol supplementation on visfatin serum levels and the insulin resistance index in obese mice. Methods: In this experimental study, 35 male NMRI mice were divided into seven groups of five: healthy control, high-fat diet, high-fat diet with HIIT, high-fat diet with EPA/DHA supplement, high-fat diet with clenbuterol supplement, high-fat diet with clenbuterol supplement and HIIT, and high-fat diet with EPA/DHA supplement and HIIT. HIIT training was conducted at 90% of maximum speed for six weeks, with five sessions per week. The EPA/DHA supplement dosage for five days of training was 180 mg, while each rat received two clenbuterol tablets in 25 cc of distilled water for the same duration. Blood samples were collected directly from the heart 48 hours after the final exercise session, centrifuged, and stored at -20°C for subsequent analysis. A specific animal kit was utilized to measure visfatin levels and the insulin resistance index. Results: The combination of six weeks of HIIT exercises with EPA/DHA and clenbuterol supplementation led to a significant reduction in visfatin levels in obese mice (P=0.001). Additionally, this regimen resulted in a notable decrease in the insulin resistance index among obese mice (P=0.001). Conclusion: Engaging in HIIT with appropriate intensity and duration can effectively reduce visfatin levels and improve insulin resistance. Furthermore, combining HIIT with the intake of supplements presents a promising approach to managing visfatin levels and insulin resistance.

ObjectiveThis study aimed to assess the risk of dietary exposure and study the residues of β-receptor agonists in livestock meat and liver marketed in Tianjin City.MethodsA total of 583 samples of pig， beef， mutton， and liver were randomly collected from supermarkets， online stores， and farmers’ markets in Tianjin City from 2016 to 2021. The β-receptor agonists clenbuterol， ractopamine， salbutamol， and terbutaline were tested by the relevant standard operating procedures of Manual China National Food Contamination and Harmful Factors Risk Monitoring， combined with residents’ food consumption data， using the index of food safety （IFS） to assess the health risk.ResultsThe overall detection rate of the 583 samples was 13.89% （81/583）. The main β-receptor agonist detected was clenbuterol， which accounted for 98.77% （80/81） of all the detected samples. Only 1 sample was detected with salbutamol， and the other 2 β-receptor agonists were not detected. The top 3 food categories in terms of detection rate were beef liver， 33.96% （36/106）； mutton， 15.79% （18/114）； and beef， 13.22% （16/121）， while the detection rate of pork and pig liver was 1.19% （1/84） and 2.00% （1/50）， respectively； the difference between the detection rates of the different categories was statistically significant （χ2=56.11， P < 0.05）. The detection rate of β-receptor agonists in samples collected from farmers’ markets was significantly higher than that collected from supermarkets and online shops （χ2= 10.91， P < 0.05）. The exposure risk assessment showed that the average exposure of pork， beef， and mutton ranged from 0.000 1 to 0.000 4 μg/kg·BW. The IFS ranged from 0.022 0 to 0.095 0， and the IFS values were less than 1.ConclusionA certain detection degree of β-receptor agonists was observed in livestock meat and liver marketed in Tianjin City， but the average exposure risk of β-receptor agonists was low from the dietary exposure of pig， beef， and mutton.

Abstract Background Alzheimer’s disease is a neurodegenerative disorder, and Aβ aggregation is considered to be the central process implicated in its pathogenesis. Current treatments are faced by challenges such as serious side effects and reduced drug bioavailability. In this study, we developed a drug delivery system for intramuscular injection that uses cellular activity to achieve constant and long-term drug release. Methods Synthesized mesoporous hydroxyapatite (SHAP) was prepared via co-precipitation, and hydrophobic surface modification using stearic acid was then used to load clenbuterol by physical absorption, thus creating the drug delivery system. Clenbuterol release was achieved through cellular activity, with macrophage uptake triggering lysosome/endosome disruption, cytoplasmic release, extracellular exocytosis, and subsequent systemic circulation. Results We found that clenbuterol-loaded SHAP enabled sustained release for more than 2 weeks and effectively modulated inflammation, reduced Aβ oligomer-induced toxicity, and prevented Aβ aggregation. Conclusions Our findings suggest that treatment with clenbuterol loaded in this SHAP delivery system could be a promising strategy for treating Alzheimer’s disease. Graphical Abstract

The abuse of clenbuterol hydrochloride (CL) is a serious threat to people's life and health. The traditional THz detection method can only detect the sample at the milligram level, which is not suitable for trace analysis of the sample. In this paper, a THz metamaterial enhanced micro-nano structure was designed based on the electromagnetic theory to realize the highly sensitive detection of the trace CL by THz spectroscopy. Firstly, four pretreatment methods were used to correct the spectrum, including AirPLS, AsLS, Backcor and BEADS. Secondly, four algorithms, namely, PCA, CARS, UVE, and SPA were used to extract features from the spectra. Finally, the PLS and LS-SVM models are established. The AirPLS-CARS-LS-SVM model has the best result, with the Rp of 0.9743, the RMSEP of 2.90 × 10–5, and the LOD of 9.67 × 10–5 mg/mL. This study will facilitate the rapid, nondestructive and highly sensitive detection of the trace CL in food. Besides, this study has significant implications for promoting the detection of other types of clenbuterol and trace substances. [ABSTRACT FROM AUTHOR]

This study demonstrates a novel colorimetric method for clenbuterol (CLB) detection based on the peroxidase-like activity of SiO2-coated Fe3O4 nanoparticles (Fe3O4@mSiO2) modified with montmorillonite (MMT) and sodium dodecyl sulfate (SDS), namely, Fe3O4@mSiO2@MMT-SDS. Fe3O4@mSiO2@MMT-SDS nanoparticles were prepared by modifying Fe3O4@mSiO2 with MMT and SDS to impart negative charges to the particle surfaces. The structural properties, steady-state kinetics, catalytic performance, and reaction conditions of the as-prepared nanoparticles were analyzed. Fe3O4@mSiO2@MMT-SDS nanoparticles exhibited excellent dispersion and enzyme-like activity, which improved the affinity with substrate (TMB and H2O2) for the catalytic oxidation of CLB by H2O2, turning the solution blue in the presence of the 3,3ʹ, 5,5ʹ-tetramethylbenzidine chromogen. Under optimal reaction conditions, a strong linear correlation between the absorbance of solution and CLB concentration (R2 = 0.9983) was observed. Here, the limit of detection, spike recovery, and relative standard deviation were 9.6 × 10-9 mol·L-1, 94.2 %–102.2 %, and 2.2 %–4.4 %, respectively. The proposed method is a simple, fast, cost-efficient, and sensitive assay, which can be used to rapidly and accurately screen CLB in pork and animal feeds.

Clenbuterol is a potent beta-2 agonist widely misused by professional athletes and bodybuilders. Information on clenbuterol associated adverse events is present in case reports and case series, though it may not be readily available. This systematic review aimed to critically evaluate the evidence of adverse events associated with clenbuterol among athletes. The search strategy was in accordance with PRISMA guidelines. Databases such as PubMed, Science Direct, Scopus, and Google Scholar were searched from 1990 to October 2021 to find out the relevant case reports and case series. There were 23 included studies. Using a suitable scale, the included studies' methodological quality analysis was evaluated. In total, 24 athletes experienced adverse events. Oral ingestion of clenbuterol was the most preferred route among them. The daily administered dose of clenbuterol was ranging from 20 µg to 30 mg. Major adverse events experienced by athletes were supraventricular tachycardia, atrial fibrillation, hypotension, chest pain, myocardial injury, myocarditis, myocardial ischemia, myocardial infarction, cardiomyopathy, hepatomegaly, hyperglycemia, and death. The cardiac-related complications were the most commonly occurring adverse events. Clenbuterol is notorious to produce life-threatening adverse events including death. Lack of evidence regarding the performance-enhancing effects of clenbuterol combined with its serious toxicities questions the usefulness of this drug in athletes. [ABSTRACT FROM AUTHOR]

Abstract Background Noradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors’ (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood. Methods Two mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD. Results Consistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity. Conclusions Our data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD.

Sepsis is a pathology accompanied by increases in myeloid cells and decreases in lymphoid cells in circulation. In a murine sepsis model induced by cecum ligation and puncture (CLP), increasing numbers of neutrophils and decreasing levels of B-cells in circulation are among the earliest changes in the immune system. However, to date, the mechanisms for these changes remain to be elucidated. The study here sought to elucidate mechanisms underlying the changes in the leukocyte levels after CLP and also to determine what, if any, role for an involvement of the sympathetic nervous system (SNS). Here, male C57/BL6 mice were subjected to CLP or sham-CLP (abdominal wall incised, but cecum was not punctured). The changes in the number of circulating leukocytes over time were then investigated using flow cytometry. The results showed that a sham-CLP led to increased polymorphonuclear cells (PMN; most of which are neutrophils) and decreased B-cells in the circulation to an extent similar to that induced by CLP. Effects of adrenergic agonists and antagonists, as well as of adrenalectomy, were also examined in mice that underwent CLP or sham-CLP. Administering adrenaline or a β2 adrenergic receptor agonist (clenbuterol) to mice 3 h before sacrifice produced almost identical changes to as what was seen 2 h after performing a sham-CLP. In contrast, giving a β2 adrenergic receptor antagonist ICI118,551 1 h before a CLP or sham-CLP suppressed the expected changes 2 h after the operations. Noradrenaline and an α1 adrenergic receptor agonist phenylephrine did not exert significant effects. Adrenalectomy 24 h before a sham-CLP significantly abolished the expected sham-CLP-induced changes seen earlier. Clenbuterol increased splenocyte expression of Cxcr4 (a chemokine receptor gene); adrenalectomy abolished sham-CLP-induced Cxcr4 expression. A CXCR4 antagonist AMD3100 repressed the sham-CLP-induced changes. From these results, it may be concluded that sepsis-induced activation of the SNS may be one cause for immune dysfunction in sepsis – regardless of the pathogenetic processes.

Clenbuterol (Clb) (4‐amino‐α‐[(tert‐butylamine) methyl]‐3,5‐dichlorobenzyl alcohol) is a sympathomimetic agent that exhibits β2‐agonist activity. It is applied as a bronchodilatory, tocolytic, and mucolytic agent and is authorized for clinical management in both human and veterinary therapeutics as a racemic mixture. However, its use is strictly prohibited in animals destined for food production in countries in the European Union and in the United States and Mexico, among many others. The R‐(−) enantiomer in clenbuterol stimulates β2‐receptors, whereas the S‐(+) enantiomer blocks the effect of β1‐receptors. The aims of this study were to develop a method for detecting and quantifying Clb and its enantiomeric distribution in several bovine tissues. The UHPLC‐MS/MS method developed to quantify the target compound at trace levels in these tissues combines high sensitivity with good selectivity and short chromatographic run time. The tissue samples tested were found to contain racemic Clb in concentrations of 5–447 pg g−1. The enantiomeric analysis of Clb showed that R‐(−)‐Clb is present at higher concentrations in some tissues, whereas S‐(+)‐Clb was detected in a ratio of 55/45 in the liver and heart tissues. [ABSTRACT FROM AUTHOR]

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2 μg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12 months at −20℃ and short-term stability for 7 days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton. [ABSTRACT FROM AUTHOR]

Background: Noradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson's disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors' (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood. Methods: Two mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD. Results: Consistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity. Conclusions: Our data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD. [ABSTRACT FROM AUTHOR]

In the present work, a nickel-ferrite/reduced-graphene-oxide composite was synthesized by a hydrothermal co-precipitation approach. The obtained materials were characterized by using X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray microscopy (mapping energy dispersive X-Ray), adsorption/desorption isotherms of nitrogen, vibrating sample magnetometer, X-ray photoelectron microscopy, and Raman spectroscopy. The obtained composite with a large surface area consists of highly dispersed nickel-ferrite nanoparticles (7–10 nm) over reduced-graphene-oxide layers. In addition, this composite possesses high magnetization saturation with a soft magnetic property. A sensitive electrochemical sensor for the determination of clenbuterol was manufactured by modifying a glassy-carbon electrode with nickel ferrite/reduced graphene oxide composite film. The peak current measured with differential pulse voltammetry for clenbuterol solutions is linearly proportional to its concentration ranging from 0.99 to 18.03 µM with a limit of detection of 0.17 µM and a limit of quantification of 0.52 µM. Furthermore, the reproducibility, precision, and accuracy of the proposed method were also investigated. This proposed method was applied to detect clenbuterol in pig urine samples with satisfactory results. [ABSTRACT FROM AUTHOR]

A rapid, simple but sensitive analytical method for the simultaneous monitoring of heavy metal ions and illegal additives in complex samples remains lacking. We reported a difunctional capture–detection surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) for the direct detection of the heavy metal residue Cd ion (Cd2+) and illegal additive clenbuterol (CLE) with high sensitivity and specificity herein. A novel magnetic SERS tag (Fe@RAu) with a Raman reporter molecule–embedded raspberry-like Au shell was fabricated via a one-step reduction reaction that not only simplified the fabrication of common magnetic SERS nanomaterials but also improved SERS activity to increase detection sensitivity. The difunctional SERS–ICA enabled the fast magnetic enrichment of the target small molecules in complex specimens and ultrasensitive and quantitative determination of CLE/Cd2+ on the two test lines of the ICA system. The limits of detection of the developed method for CLE and Cd2+ were as low as 0.48 and 1.88 pg/mL, respectively, and about 2000 and 500 times lower than those of conventional AuNP-based ICA strips. The excellent practicability of Fe@RAu–ICA for real food samples analysis was also demonstrated, revealing the potential of the proposed assay for rapid and on-site screening of small-molecule pollutants. [Display omitted] • A Raman reporter molecule–embedded magnetic SERS tag was reported for the first time. • Raman reporter–embedded magnetic tag was used as capture/detection difunctional tool for small molecule detection. • Simultaneous monitoring of heavy metal ions and illegal additives on SERS–ICA was first realized. • The detection limits for CLE and Cd2+ were as low as 0.48 and 1.88 pg/mL, respectively. [ABSTRACT FROM AUTHOR]

Clenbuterol (CLB) is a bronchodilator for treating respiratory disorders. However, the irrational use of CLB in animal feeding can cause serious risks to human health. The development of selective and efficient adsorbent for CLB removal is of great significance. Herein, we report a convenient covalent grafting approach for the fabrication of molecularly imprinted covalent organic framework (COF) for selective adsorption and removal of CLB from environmental water and milk samples. By integrating the merits of both molecularly imprinted polymers (MIPs) and COFs, the molecularly imprinted COFs named BatBt-MIPs (2,5-bis(allyloxy)terephthalohydrazide, Bat; 1,3,5-benzenetricarboxaldehyde, Bt) presented the alluring traits of large surface area, tailored-made binding sites, uniform pore structure, and good selectivity for CLB. The adsorption equilibrium for CLB on BatBt-MIPs was quickly reached within 20 min and followed pseudo-second-order kinetic model. BatBt-MIPs showed a CLB adsorption capacity of 15.45 mg/g, which is about 3.7 times higher than that of non-imprinted COFs (BatBt-NIPs). Compared with BatBt-NIPs, BatBt-MIPs present improved adsorption selectivity for template CLB. Selective removal of CLB from complex environmental water and milk samples on BatBt-MIPs was also realized. This work provided a new covalent grafting approach to construct novel molecularly imprinted COFs and highlighted the great potential of COF-MIPs in contaminants elimination. [Display omitted] • Novel molecularly imprinted BatBt-MIPs was prepared via covalent graft strategy. • BatBt-MIPs exhibited good selectivity for the clenbuterol adsorption and removal. • BatBt-MIPs could effectively remove clenbuterol from water and milk sample. [ABSTRACT FROM AUTHOR]

[Display omitted] • The CuO@Ag composite was synthesized by metal self-reducing triggered green etching. • Flower-like morphology of the CuO@Ag improved its binding ability with antibodies. • The CuO@Ag has excellent photothermal conversion efficiency. • The dual-mode immunosensor can achieve high-sensitive detection of clenbuterol. Photothermal materials has aroused extensive attention for building multi-signal immunosensors. Herein, a metal self-reducing triggered green etching strategy was employed for the synthesis of CuO@Ag nanoparticles (NPs) with high photothermal conversion efficiency and better antibody coupling efficiency. The flower-like morphology of the CuO@Ag NPs has improved its binding ability with antibodies, which enables them to serve as the multifunctional signal label of the lateral flow immunoassay (LFIA). Then, a colorimetric-photothermal dual-mode LFIA (CuO@Ag-based LFIA) was designed for the highly sensitive detection of clenbuterol hydrochloride (CLE). After direct, the CuO@Ag NPs endowed T-line to be irradiated by an 808 nm laser to obtain photothermal signal owing to their strong photothermal conversion efficiency of CuO@Ag. Under optimal conditions, the detection limits of the CuO@Ag-based LFIA are 0.205 ng mL−1 and 0.131 ng mL−1 under colorimetric and photothermal modes, respectively, which are 13 and 20 times sensitive than that of the conventional AuNP-based LFIA. Additionally, the LFIA was assessed by measuring CLE in food samples with recovery rates ranging from 86.37 % to 103.72 % and coefficient of variations (CVs) ranging from 2.52 % to 9.64 %. Consequently, this work provides qualitative-quantitative point-of-care testing (POCT) approach and has important reference value for other facile, green, and low-cost synthesis of composite signal labels in the LFIA system. [ABSTRACT FROM AUTHOR]

The World Anti-Doping Agency (WADA) has prohibited the use of clenbuterol (CLN) because it induces anabolic muscle growth while potentially causing adverse effects such as palpitations, anxiety, and muscle tremors. Thus, it is vital to assess meat quality because, athletes might have positive test for CLN even after consuming very low quantity of CLN contaminated meat. Numerous materials applied for CLN monitoring faced potential challenges like sluggish ion transport, non-uniform ion/molecule movement, and inadequate electrode surface binding. To overcome these shortcomings, herein we engineered bimetallic zeolitic imidazole framework (BM-ZIF) derived N-doped porous carbon embedded Co nanoparticles (CN-CoNPs), dispersed on conductive cellulose acetate-polyaniline (CP) electrospun nanofibers for sensitive electrochemical monitoring of CLN. Interestingly, the smartly designed CN-CoNPs wrapped CP (CN-CoNPs-CP) electrospun nanofibers offers rapid diffusion of CLN molecules to the sensing interface through amine and imine groups of CP, thus minimizing the inhomogeneous ion transportation and inadequate electrode surface binding. Additionally, to synchronize experiments, machine learning (ML) algorithms were applied to optimize, predict, and validate voltametric current responses. The ML-trained sensor demonstrated high selectivity, even amidst interfering substances, with notable sensitivity (4.7527 μA/μM/cm2), a broad linear range (0.002–8 μM), and a low limit of detection (1.14 nM). Furthermore, the electrode exhibited robust stability, retaining 98.07% of its initial current over a 12-h period. This ML-powered sensing approach was successfully employed to evaluate meat quality in terms of CLN level. To the best of our knowledge, this is the first study of using ML powered system for electrochemical sensing of CLN. [ABSTRACT FROM AUTHOR]

Abstract Background Unregulated use of a variety of drugs and supplements by bodybuilders and athletes is common and can lead to severe adverse complications. Only a small proportion of acute pancreatitis cases are drug induced, and case reports are essential for identifying potential drug-related risks for pancreatitis. Here we present the first case report published of acute pancreatitis linked to recreational use of anabolic–androgenic steroids, subcutaneous growth hormone, and clenbuterol in a previously healthy male after excluding all other causes of pancreatitis. Case presentation A 31-year-old Arab male bodybuilder presented with acute abdominal pain associated with nausea and sharp pain radiating to the back. The patient was not using tobacco or alcohol but was using multiple drugs related to bodybuilding, including anabolic–androgenic steroids, subcutaneous growth hormone, clenbuterol, and multiple vitamin supplements. Laboratory studies revealed a normal white blood cell count, elevated C-reactive protein, minimally elevated aspartate aminotransferase and total bilirubin with normal remaining liver tests, and elevated amylase and lipase. The patient had no hypertriglyceridemia or hypercalcemia, and had had no recent infections, abdominal procedures, trauma, or scorpion exposure. Imaging and laboratory investigations were negative for biliary disease and IgG4 disease. Abdominal computed tomography revealed hepatomegaly and diffuse thickening and edema of the body and tail of the pancreas with peripancreatic fat stranding. An abdominal ultrasound showed slight hepatomegaly with no evidence of cholelithiasis. Genetic testing for hereditary pancreatitis-related mutations was negative. A diagnosis of drug-induced acute pancreatitis was made, and he was treated with aggressive intravenous hydration and pain management. The patient has avoided further use of these drugs and supplements and had no further episodes of pancreatitis during 1 year of follow-up. Conclusions This case describes a patient with drug-induced acute pancreatitis after the intake of anabolic–androgenic steroids, subcutaneous growth hormone, and clenbuterol, where all other common causes of acute pancreatitis were excluded. Clinicians should be alert to the possibility of drug-induced acute pancreatitis occurring in bodybuilders and athletes using similar drug combinations.

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, these requirements were met by a two-dimensional separation method based on online solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (SPE–UHPLC–MS/MS). The developed method provides favorable performance parameters, and it is characterized by minimum manual steps (only dilution and the addition of an internal standard) in the sample preparation. A limit of quantification (LOQ) of 0.1 ng/mL, excellent linearity (0.9999), remarkable precision (1.26% to 8.99%) and high accuracy (93.1% to 98.7%) were achieved. From a practical point of view, the analytical performance of the validated SPE–UHPLC–MS/MS method was demonstrated on blinded spiked urine samples from ten healthy volunteers. The estimated concentrations of clenbuterol were in accordance with their corresponding nominal values, as supported by the precision and accuracy data (relative standard deviation ≤5.4%, relative error ≤11%). The fulfillment of the World Anti-Doping Agency's screening and confirmation criteria indicates that the proposed method is suitable for implementation in routine use in toxicologic and antidoping laboratories. Due to its high orthogonality and separation efficiency, the SPE–UHPLC–MS/MS method should also be easily adapted to the separation of structurally related compounds (such as clenbuterol metabolites). Thus, future antidoping applications could also include monitoring of clenbuterol metabolites, providing a longer detection widow. [ABSTRACT FROM AUTHOR]

Sepsis is a pathology accompanied by increases in myeloid cells and decreases in lymphoid cells in circulation. In a murine sepsis model induced by cecum ligation and puncture (CLP), increasing numbers of neutrophils and decreasing levels of B-cells in circulation are among the earliest changes in the immune system. However, to date, the mechanisms for these changes remain to be elucidated. The study here sought to elucidate mechanisms underlying the changes in the leukocyte levels after CLP and also to determine what, if any, role for an involvement of the sympathetic nervous system (SNS). Here, male C57/BL6 mice were subjected to CLP or sham-CLP (abdominal wall incised, but cecum was not punctured). The changes in the number of circulating leukocytes over time were then investigated using flow cytometry. The results showed that a sham-CLP led to increased polymorphonuclear cells (PMN; most of which are neutrophils) and decreased B-cells in the circulation to an extent similar to that induced by CLP. Effects of adrenergic agonists and antagonists, as well as of adrenalectomy, were also examined in mice that underwent CLP or sham-CLP. Administering adrenaline or a β2 adrenergic receptor agonist (clenbuterol) to mice 3 h before sacrifice produced almost identical changes to as what was seen 2 h after performing a sham-CLP. In contrast, giving a β2 adrenergic receptor antagonist ICI118,551 1 h before a CLP or sham-CLP suppressed the expected changes 2 h after the operations. Noradrenaline and an α1 adrenergic receptor agonist phenylephrine did not exert significant effects. Adrenalectomy 24 h before a sham-CLP significantly abolished the expected sham-CLP-induced changes seen earlier. Clenbuterol increased splenocyte expression of Cxcr4 (a chemokine receptor gene); adrenalectomy abolished sham-CLP-induced Cxcr4 expression. A CXCR4 antagonist AMD3100 repressed the sham-CLP-induced changes. From these results, it may be concluded that sepsis-induced activation of the SNS may be one cause for immune dysfunction in sepsis – regardless of the pathogenetic processes. [ABSTRACT FROM AUTHOR]

β2‐adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri‐TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro‐dose of 2.5 μg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml. [ABSTRACT FROM AUTHOR]

The illegal use of clenbuterol seriously endangers food safety and human health. Accurate monitoring of the illegal use of clenbuterol in livestock can efficiently prevent the clenbuterol residue pork products from entering the consumer market. Thus, in this study, a simple, rapid, and sensitive method for the determination of clenbuterol in swine urine was developed using electromembrane extraction combined with liquid chromatography‐tandem mass spectrometry. It should be noted that the electromembrane extraction method presented many advantages of simple operation, fast mass transfer rate, good sample clean‐up capability, and less organic solvent consumption. The effect of important factors on the extraction efficiency of clenbuterol was investigated. Under the optimal conditions, good linearity was achieved for clenbuterol over the range of 1–1000 ng/ml (linear correlation [R2] = 0.9996). The recoveries of clenbuterol in swine urine at three spiked levels ranged from 83.7% to 110.0% with relative standard deviation values lower than 9.7% (n = 4). The limits of detection and quantification for clenbuterol were 0.07 and 0.25 ng/ml, respectively. These results suggested that the proposed method has great potential for the extraction and determination of trace analyte in a complex sample matrix for monitoring illegal use in livestock. [ABSTRACT FROM AUTHOR]

• Clenbuterol (CBL) suppressed post-lipopolysaccharide (LPS) ex vivo immune activation. • IL-1β, IL-6, and TNF-α were among the cytokines most substantially attenuated by CBL. • Anticipatory and consummatory anhedonia correlated with a CBL-treated immune cluster composing predominantly of hematopoietic growth factors. • β2-agonism might serve a protective role in adolescent affective symptomatology. While inflammation has been implicated in psychopathology, relationships between immune-suppressing processes and psychiatric constructs remain elusive. This study sought to assess whether β 2 -agonist clenbuterol (CBL) would attenuate immune activation in adolescents with mood and anxiety symptoms following ex vivo exposure of whole blood to lipopolysaccharide (LPS). Our focus on adolescents aimed to target a critical developmental period when psychiatric conditions often emerge and prior to chronicity effects. To capture a diverse range of immunologic and symptomatologic phenotypes, we included 97 psychotropic-medication free adolescents with mood and anxiety symptoms and 33 healthy controls. All participants had comprehensive evaluations and dimensional assessments of psychiatric symptoms. Fasting whole-blood samples were collected and stimulated with LPS in the presence and absence of CBL for 6 hours, then analyzed for 41 cytokines, chemokines, and hematopoietic growth factors. Comparison analyses used Bonferroni-corrected nonparametric tests. Levels of nine immune biomarkers—including IL-1RA, IL-1β, IL-6, IP-10, MCP-1, MIP-1α, MIP-1β, TGF-α, and TNF-α—were significantly reduced by CBL treatment compared to LPS alone. Exploratory factor analysis reduced 41 analytes into 5 immune factors in each experimental condition, and their relationships with psychiatric symptoms were examined as a secondary aim. CBL + LPS Factor 4—comprising EGF, PDGF-AA, PDGF-AB/BB, sCD40L, and GRO—significantly correlated with anticipatory and consummatory anhedonia, even after controlling for depression severity. This study supports the possible inhibitory effect of CBL on immune activation. Using a data-driven method, distinctive relationships between CBL-affected immune biomarkers and dimensional anhedonia were reported, further elucidating the role of β 2 -agonism in adolescent affective symptomatology. [ABSTRACT FROM AUTHOR]

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic β2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs. In this study, the authors demonstrated that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling may prove beneficial as novel antidiabetic drugs. [ABSTRACT FROM AUTHOR]

Clenbuterol (CB) is a synthetic β-receptor agonist which can be used to improve carcass leanness in swine, but its residues in pork also pose health risks. In this report, surface-enhanced Raman scattering (SERS) technology was used to achieve rapid detection and identification of clenbuterol hydrochloride (CB) residues. First, the effects of several different organic solvents on the extraction efficiency were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Then, SERS signals of clenbuterol in different solvents were compared, and it was found that clenbuterol had a better enhancement effect in an aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Next, enhancement effect was compared using different concentration of sodium chloride solution as the aggregating compound. Finally, pork samples with different clenbuterol content (1, 3, 5, 7, 9, and 10 µg/g) were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 mol/L of NaCl solution as aggregating compound and gold colloid as an enhanced substrate. Multiple scattering correction (MSC) and automatic Whittaker filter (AWF) were used for preprocessing, and the fluorescence background contained in the original Raman spectra was removed. A unary linear regression model was established between SERS intensity at 1472 cm-1 and clenbuterol content in pork samples. The model had a better linear relationship with a correlation coefficient R2 of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid screening of pork containing clenbuterol in the market. [ABSTRACT FROM AUTHOR]

The residue of clenbuterol (CLB) in food poses a potential harm to human health. Herein, we presented an electrochemical aptasensor (E-A-CRISPR) based on employing an aptamer as a specific recognition element and asymmetric CRISPR-Cas12a as signal amplifiers for sensitive, and selective detection of CLB. In this E-A-CRISPR system, the target CLB bound to the aptamer and initiated cascade signal amplification through the DNase activity of CRISPR-Cas12a with two competitive gRNAs. Upon amplification, the active Cas12a cleaved the methylene blue-labeled hairpin probe on the electrode, reducing the peak current. Under optimal conditions, the E-A-CRISPR system showed a wide linear range (1 pM-100 nM) and a low detection limit (500 fM). This system could detect CLB in potable water, pig liver, and pork samples, showing significant potential for food safety monitoring. To our knowledge, this study is the first to use a CRISPR-Cas12a powered system for electrochemical sensing of CLB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sports doping remains a significant challenge in competitive sports. Given that urine analysis is the standard for detecting doping, developing rapid, sensitive, accurate, and high-throughput methods for stimulant detection in urine is crucial. Surface-enhanced Raman scattering (SERS) tag-based immunoassays have emerged as powerful analytical tools known for their high sensitivity and specificity, holding particular promise for stimulant detection in urine samples. However, both the Raman signals of typical SERS tags and sample matrices are within the Raman fingerprint region (<1800 cm -1 ), which could lead to spectrum overlap, potentially reducing detection accuracy and sensitivity. By recognizing this, we designed a competitive immunoassay that integrates two types of zero-background SERS tags and magnetic separation. These innovative SERS tags exhibit distinctive Raman peaks within the Raman-silent region (1800-2800 cm -1 ), effectively mitigating potential spectrum overlap with background sample signals. Moreover, magnetic separation not only enhances operational simplicity but also improves the system's anti-interference capability. Using clenbuterol (CL) and higenamine (HM) as model targets, the SERS-based competitive immunoassay demonstrated sensitive detection of individual CL or HM standards, with limits of detection (LODs) of 0.87 and 0.71 pg/mL, respectively. In multiplex mode, CL and HM can be simultaneously detected with LODs of 1.0 and 0.81 pg/mL, respectively. Furthermore, the recovery rates in urine samples ranged from 83 to 116% (relative standard deviation, RSD ≤ 6.4%) for CL and from 82 to 103% (RSD ≤ 5.1%) for HM, further confirming the reliability of the SERS-based immunoassay for practical applications.

In this study, a fluorescence detection method combining aptamer-modified up-conversion nanoparticles (UCNPs) and magnetic nanoparticles (MNPs) was developed for detection of Clenbuterol (CLB). The aptamer-modified magnetic NPs captured CLB, which reacted with the aptamer-modified UCNPs and generated a sandwich complex. The aptamer-modified UCNPs acted as a fluorescence source. The MNP-CLB-UCNP complex was retrieved from the solution using an magnetic field, and the fluorescence intensity was detected by fluorescence spectrophotometry with excitation and emission spectra at 980 nm and in the 400-800 nm region, respectively. The results showed that the fluorescence intensity gradually increased with increasing concentrations of CLB with a good specify. The method was highly sensitive for the quantification of CLB, with a limit of detection of 0.304 ng mL -1 . The recovery rate of CLB from pork samples ranged from 84 % to 94.87 %. This fluorescence method enables the sensitive, precise, and accurate quantification of CLB residues in pork samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Aims: Cerebral hypometabolism occurs years prior to a diagnosis of neurodegenerative diseases and coincides with reduced cerebral perfusion and declining noradrenergic transmission from the locus coeruleus. In pre-clinical models, β-adrenoceptor (β-AR) agonists increase cerebrocortical glucose metabolism, and may have therapeutic potential for neurodegenerative diseases. This study investigated the safety and effects on regional cerebral blood flow (rCBF) of the oral, brain-penetrant β 2 -AR agonist, clenbuterol, in healthy volunteers (HV) and patients with mild cognitive impairment (MCI) or Parkinson's disease (PD)., Methods: This study evaluated the safety and effects on cerebral activity of the oral, brain-penetrant, β 2 -AR agonist clenbuterol (20-160 μg) in healthy volunteers and patients with MCI or PD. Regional CBF, which is tightly coupled to glucose metabolism, was measured by arterial spin labelling MRI in 32 subjects (25 HV and 8 MCI or PD) across five cohorts. In some cohorts, low doses of nadolol (1-5 mg), a β-AR antagonist with minimal brain penetration, were administered with clenbuterol to control peripheral β 2 -AR responses., Results: Significant, dose-dependent increases in rCBF were seen in multiple brain regions, including hippocampus, amygdala and thalamus, following the administration of clenbuterol to HVs (mean changes from baseline in hippocampal rCBF of -1.7%, 7.3%, 22.9%, 28.4% 3 h after 20, 40, 80 and 160 μg clenbuterol, respectively). In patients with MCI or PD, increases in rCBF following 80 μg clenbuterol were observed both without and with 5 mg nadolol (in hippocampus, 18.6%/13.7% without/with nadolol). Clenbuterol was safe and well-tolerated in all subjects; known side effects of β 2 -agonists, including increased heart rate and tremor, were mild in intensity and were blocked by low-dose nadolol., Conclusions: The effects of clenbuterol on rCBF were evident both in the absence and presence of low-dose nadolol, suggesting central nervous system (CNS) involvement. Concomitant inhibition of the peripheral effects of clenbuterol by nadolol confirms that meaningful β 2 -AR antagonism in the periphery was achieved without interrupting the central effects of clenbuterol on rCBF., (© 2024 British Pharmacological Society.)

In the present work, tungsten disulfide was synthesised and characterised by using X-ray diffraction and scanning electron microscopy. This material was employed to modify a glassy-carbon electrode to analyse clenbuterol. The results show that tungsten disulfide displays electrocatalytic activity toward clenbuterol oxidation. The proposed technique is prospective for clenbuterol examination. The limit of detection and the linear range are 0.51 mM and 1–210 mM.

A label-free fluorescent biosensor based on the clenbuterol binding aptamer and the fluorescent intercalator SYBR Green I (SGI) was established for the sensitive and selective detection of clenbuterol. In the absence of clenbuterol, a weak fluorescence signal of SGI was observed. In the presence of clenbuterol, the specific recognition and high-affinity binding of the aptamer with clenbuterol induced a conformational change of the aptamer, and a longer double-stranded DNA was formed. As a result, an enhancement in the fluorescence of SGI could be found. Under optimal conditions, the fluorescence change ratio increased linearly with clenbuterol concentration from 50 nM to 450 nM, with a detection limit of 18.8 nM. In addition, the proposed aptasensor showed good selectivity and was successfully applied for clenbuterol quantification in pork samples with recoveries of 90.37–104.98%. [ABSTRACT FROM AUTHOR]

Direct and sensitive detection of multiple illegal additives in complex food samples is still a challenge in on-site detection. In this study, an ultrasensitive immunochromatographic assay (ICA) using magnetic Fe3O4@Au nanotags as a capture/detection difunctional tool was developed for the direct detection of β2-adrenoceptor agonists in real samples. The Fe3O4@Au tag is composed of a large magnetic core (~160 nm), a rough Au nanoshell, dense surface-modified Raman molecules, and antibodies, which cannot only effectively enrich targets from complex solutions to reduce the matrix effects of food samples and improve detection sensitivity, but also provide strong colorimetric/surface-enhanced Raman scattering (SERS) dual signals for ICA testing. The dual readout signals of the proposed ICA can meet the detection requirements in different environments. Specifically, the colorimetric signal allows for rapid visual detection of the analyte, and the SERS signal is used for the sensitive and quantitative detection modes. The proposed dual-signal ICA can achieve the simultaneous determination of two illegal additives, namely, clenbuterol hydrochloride and ractopamine. The detection limits for the two targets via colorimetric and SERS signals were down to ng mL−1 and pg mL−1 levels, respectively. Moreover, the proposed assay has demonstrated high accuracy and stability in real food samples. [ABSTRACT FROM AUTHOR]

Simple Summary: Although β-receptor agonists are tightly controlled, there is still abuse. A general high-performance liquid chromatography-tandem mass spectrometry detection method was developed to measure three typical β-receptor agonists; the metabolites and metabolic pathways of SAL, CLB, and RAC in rats were investigated. The results showed that the metabolites in the rats mainly existed in the form of the prototype drugs and that they still had a few metabolites. The absorption and metabolism of the different β-receptor agonists in the rats were quite different, and the absorption and metabolism of the same β-receptor agonists in different tissues were also different. This will help to modulate the receptor agonist and to study its detection and metabolism in other species. This paper developed a universal detection method by high-performance liquid chromatography-tandem mass spectrometry to detect three typical clenbuterols, CLB, SAL, and RAC, and to investigate the metabolism of β-agonists in vivo. The parent ions and daughter ions of the three β-receptor agonist standards and the residues in the muscle, liver, and blood samples of rats were obtained by Total Ions Scan mode. The metabolites produced in different tissues at a specific time were qualitatively and quantitatively analyzed, and the corresponding metabolic pathways were inferred. The results showed that the three β-receptor agonists mainly existed in the form of prototype drugs in rats, with a small amount of clenbuterol methyl compound and albuterol methyl compound. There were significant differences in residual metabolism between different tissues of the same species. In addition, different β-receptor agonists have different absorption and utilization rates in rats. [ABSTRACT FROM AUTHOR]

Abstract Background and objective Sympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair. Materials and methods To evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin–eosin were used to evaluate SCs, myogenic repair and differentiation. Results The number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB. Conclusion Normal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

The residue of clenbuterol (CLB) in food poses a potential harm to human health. Herein, we presented an electrochemical aptasensor (E -A-CRISPR) based on employing an aptamer as a specific recognition element and asymmetric CRISPR-Cas12a as signal amplifiers for sensitive, and selective detection of CLB. In this E -A-CRISPR system, the target CLB bound to the aptamer and initiated cascade signal amplification through the DNase activity of CRISPR-Cas12a with two competitive gRNAs. Upon amplification, the active Cas12a cleaved the methylene blue-labeled hairpin probe on the electrode, reducing the peak current. Under optimal conditions, the E -A-CRISPR system showed a wide linear range (1 pM-100 nM) and a low detection limit (500 fM). This system could detect CLB in potable water, pig liver, and pork samples, showing significant potential for food safety monitoring. To our knowledge, this study is the first to use a CRISPR-Cas12a powered system for electrochemical sensing of CLB. • CRISPR-Cas12a coupled with an aptamer can achieve selective detection of clenbuterol (CLB). • Integrating asymmetric CRISPR-Cas12a with electrochemistry to enhance sensitivity • High selectivity and sensitivity detection of CLB in a linear range from 1 pM to 100 nM with a detection limit of 500 fM. • Reliable detection of CLB in potable water, pig liver, and pork samples was achieved. [ABSTRACT FROM AUTHOR]

To achieve high throughput detection of clenbuterol while reducing matrix interference, we developed a simple and integrated nano-inspection platform. Herein, based on the simplicity and convenience of paper chip, a multi-layer paper chip-chemiluminescence (CL) sensor was designed in the form of "48-well plate" type. The all-in-one paper chip-CL sensor has integrated the covalent organic frameworks (COFs) layer for sample matrix purification, high affinity nanobody layer for specific capture of target, and CL rapid response layer for the final determination. The signal readout was realized by the inhibitory effect of clenbuterol on the K 3 [Fe(CN) 6 ] CL system. Detection of clenbuterol in pork meat was verified by measuring the CL intensity and satisfactory recoveries (85.9 %–97.3 %) were achieved. 48 samples could be simultaneously detected on one-chip in one-test, favoring the high-throughput detection. Especially, the work explored a new pathway for design of portable analytical instruments to achieve immediate on-site detection of hazards. Multi-layer paper chip sensing platform was established by COF sample pretreatment, nanobody recognition, and luminescent sensing for high-throughput and rapid nano-inspection of food hazards. [Display omitted] • A new paper chip method offers low-cost, easy-to-use, efficient clenbuterol detection. • Novel COF layer contributed to matrix cleanup for complex samples. • High affinity nanobody layer ensured high selectivity in CL detection. [ABSTRACT FROM AUTHOR]