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Amitraz (AMT) is a broad-spectrum formamidine insecticide and acaricide. In this study, we produced an anti-AMT monoclonal antibody (mAb) with high performance. The half-maximal inhibitory concentration of the anti-AMT mAb was 4.418 ng/mL, the cross reactivity with other insecticides was negligible, and an affinity constant was 2.06 × 10 9 mmol/L. Additionally, we developed an immunochromatographic assay for the rapid detection of AMT residues in oranges, tomatoes, and eggplants. The cut-off values were 2000 μg/kg in oranges and tomato samples and 1000 μg/kg in eggplant samples and the calculated limits of detection were 14.521 μg/kg, 6.281 μg/kg, and 3.518 μg/kg in oranges, tomatoes, and eggplants, respectively, meeting the detection requirements for AMT in fruits and vegetables. The recovery rates ranged between 95.8 % and 105.2 %, consistent with the recovery rates obtained via LC-MS/MS. Our developed immunochromatographic assay can effectively, accurately, and rapidly determine AMT residues in oranges, tomatoes, and eggplants., 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.)

In recent years, there has been growing concern on the potential weakening of honey bees and their increased susceptibility to pathogens due to chronic exposure to xenobiotics. The present work aimed to study the effects on bees undergoing an infection by Nosema ceranae and being exposed to a frequently used in-hive acaricide, amitraz. To achieve this, newly emerged bees were individually infected with N. ceranae spores and/or received a sublethal concentration of amitraz in their diets under laboratory conditions. Mortality, food intake, total volume excrement, body appearance, and parasite development were registered. Bees exposed to both stressors jointly had higher mortality rates compared to bees exposed separately, with no difference in the parasite development. An increase in sugar syrup consumption was observed for all treated bees while infected bees fed with amitraz also showed a diminishment in pollen intake. These results coupled with an increase in the total number of excretion events, alterations in behavior and body surface on individuals that received amitraz could evidence the detrimental action of this molecule. To corroborate these findings under semi-field conditions, worker bees were artificially infected, marked, and released into colonies. Then, they were exposed to a commercial amitraz-based product by contact. The recovered bees showed no differences in the parasite development due to amitraz exposure. This study provides evidence to which extent a honey bee infected with N. ceranae could potentially be weakened by chronic exposure to amitraz treatment., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The aim of this work was to develop a fast, simple, and reliable UPLC-MS 3 method for the sensitive detection of acetochlor in biological samples. In MS 3 mode, the ion transition m/z 270.1 → 224.1→148.1 was chosen for quantification with butachlor as the internal standard. In the UPLC system, separation was performed on a UPLC column (2.1 × 50 mm ID, 1.7 μm) with 0.1 % FA in water and acetonitrile as mobile phases. After simple protein precipitation via acetonitrile, the method was well validated with good linearity (0.5-20 ng/mL, r > 0.995), accuracy (-3.70 %-2.98 %), and precision (<15 %). The selectivity and sensitivity were improved obviously in MS 3 mode than that in MRM mode. The developed UPLC-MS 3 method was successfully applied to the cellular pharmacokinetics study of acetochlor in MCF-7 cells., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024. Published by Elsevier Inc.)

A cutting-edge electrochemical method is presented for precise quantification of amitraz (AMZ), a commonly used acaricide in veterinary medicine and agriculture. Leveraging a lab-made screen-printed carbon electrode modified with a synergistic blend of perylene tetracarboxylic acid (PTCA), mesoporous carbon (MC), and Nafion, the sensor's sensitivity was significantly improved. Fine-tuning of PTCA, MC, and Nafion ratios, alongside optimization of the pH of the supporting electrolyte and accumulation time, resulted in remarkable sensitivity enhancements. The sensor exhibited a linear response within the concentration range 0.01 to 0.70 μg mL -1 , boasting an exceptionally low limit of detection of 0.002 μg mL -1 and a limit of quantification of 0.10 μg mL -1 , surpassing maximum residue levels permitted in honey, tomato, and longan samples. Validation with real samples demonstrated high recoveries ranging from 80.8 to 104.8%, with a relative standard deviation below 10%, affirming the method's robustness and precision. The modified PTCA/MC/Nafion@SPCE-based electrochemical sensor not only offers superior sensitivity but also simplicity and cost-effectiveness, making it a pivotal tool for accurate AMZ detection in food samples. Furthermore, beyond the scope of this study, the sensor presents promising prospects for wider application across various electrochemical analytical fields, thereby significantly contributing to food safety and advancing agricultural practices., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The blood-brain barrier (BBB) and tight junction (TJ) proteins maintain the homeostasis of the central nervous system (CNS). The dysfunction of BBB allows peripheral T cells infiltration into CNS and contributes to the pathophysiology of multiple sclerosis (MS). Teriflunomide is an approved drug for the treatment of MS by suppressing lymphocytes proliferation. However, whether teriflunomide has a protective effect on BBB in MS is not understood. We found that teriflunomide restored the injured BBB in the EAE model. Furthermore, teriflunomide treatment over 6 months improved BBB permeability and reduced peripheral leakage of CNS proteins in MS patients. Teriflunomide increased human brain microvascular endothelial cell (HBMEC) viability and promoted BBB integrity in an in vitro cell model. The TJ protein claudin-1 was upregulated by teriflunomide and responsible for the protective effect on BBB. Furthermore, RNA sequencing revealed that the Wnt signaling pathway was affected by teriflunomide. The activation of Wnt signaling pathway increased claudin-1 expression and reduced BBB damage in cell model and EAE rats. Our study demonstrated that teriflunomide upregulated the expression of the tight junction protein claudin-1 in endothelial cells and promoted the integrity of BBB through Wnt signaling pathway., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Researchers are increasingly using insights derived from large-scale, electronic healthcare data to inform drug development and provide human validation of novel treatment pathways and aid in drug repurposing/repositioning. The objective of this study was to determine whether treatment of patients with multiple sclerosis with dimethyl fumarate, an activator of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, results in a change in incidence of type 2 diabetes and its complications. This retrospective cohort study used administrative claims data to derive four cohorts of adults with multiple sclerosis initiating dimethyl fumarate, teriflunomide, glatiramer acetate or fingolimod between January 2013 and December 2018. A causal inference frequentist model averaging framework based on machine learning was used to compare the time to first occurrence of a composite endpoint of type 2 diabetes, cardiovascular disease or chronic kidney disease, as well as each individual outcome, across the four treatment cohorts. There was a statistically significantly lower risk of incidence for dimethyl fumarate versus teriflunomide for the composite endpoint (restricted hazard ratio [95% confidence interval] 0.70 [0.55, 0.90]) and type 2 diabetes (0.65 [0.49, 0.98]), myocardial infarction (0.59 [0.35, 0.97]) and chronic kidney disease (0.52 [0.28, 0.86]). No differences for other individual outcomes or for dimethyl fumarate versus the other two cohorts were observed. This study effectively demonstrated the use of an innovative statistical methodology to test a clinical hypothesis using real-world data to perform early target validation for drug discovery. Although there was a trend among patients treated with dimethyl fumarate towards a decreased incidence of type 2 diabetes, cardiovascular disease and chronic kidney disease relative to other disease-modifying therapies-which was statistically significant for the comparison with teriflunomide-this study did not definitively support the hypothesis that Nrf2 activation provided additional metabolic disease benefit in patients with multiple sclerosis., Competing Interests: This work was funded by Eli Lilly and Company and all authors are employees of Eli Lilly and Company. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Alan J.M. Brnabic was involved with the conceptualization, methodology, investigation and formal analysis of the data for the work and contributed to the original draft preparation, review and editing of the manuscript. Sarah E. Curtis was involved with the conceptualization, methodology, investigation and formal analysis of the data for the work and contributed to the review and editing of the manuscript. Joseph A. Johnston was involved with the conceptualization, methodology and investigation of the data for the work, and contributed to the original draft preparation, review and editing of the manuscript. Albert contributed to the review and editing of the manuscript. Anthony J. Zagar was involved with the methodology and investigation of the data for the work and contributed to the original draft preparation of the manuscript. Ilya Lipkovich was involved with the methodology and validation of the data for the work and contributed to the original draft preparation of the manuscript. Zbigniew Kadziola was involved with the formal analysis of the data for the work and contributed to the review and editing of the manuscript. Megan H. Murray was involved with the investigation, methodology and formal analysis of the data for the work and contributed to the original draft preparation of the manuscript. Timothy Ryan was involved with the conceptualization and investigation of the data for the work and contributed to the original draft preparation, review and editing of the manuscript. All authors have participated sufficiently in the work to agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors give final approval of the manuscript to be published., (Copyright: © 2024 Brnabic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Teriflunomide is administered orally to treat relapsing-remitting multiple sclerosis. In this prospective pilot study, the free and total serum concentrations of teriflunomide obtained during routine health care were measured and their relationship with disease activity was evaluated., Methods: Eighty-nine patients were included in this study. Blood samples were collected from April 2021 to February 2022, and free and total teriflunomide serum concentrations were measured. Patient assessment involved monitoring of blood counts and potential adverse effects of teriflunomide., Results: In the steady-state group, total teriflunomide concentrations ranged from 14.7 to 144.2 mg/L, while free concentrations from 31.1 to 389.7 μg/L. In the non-steady-state group, the total concentration ranged from 2.2 to 59.3 mg/L, with free concentrations ranging from 6.8 to 143.5 μg/L. In the steady-state group, a significant inverse correlation was found between absolute peripheral blood lymphocyte count and both total and free teriflunomide serum concentrations., Conclusion: Although all patients were treated with the same dose, up to a 10-fold difference in total and free teriflunomide serum concentrations, and up to a 5-fold difference in steady-state trough concentrations were observed. This vast interindividual variability can potentially lead to toxicity or, conversely, to suboptimal therapeutic concentrations of teriflunomide, with the risk of further worsening of multiple sclerosis compensation., Competing Interests: Declaration of competing interest All authors advise that there are not any actual or potential financial or other conflict of interest related to the submitted manuscript (ownership, equity position, stock options, consulting fees, patent rights, and corporate affiliations) associated with any drug, product, process, or commercial laboratory mentioned in the submitted material., (Copyright © 2023. Published by Elsevier B.V.)

Objectives: The aims of this study were to report the effectiveness and safety of teriflunomide in Chinese patients with relapsing-remitting multiple sclerosis (RRMS) and to explore the association of paramagnetic rim lesion (PRL) burden with patient outcome in the context of teriflunomide treatment and the impact of teriflunomide on PRL burden., Methods: This is a prospective observational study. A total of 100 RRMS patients treated with teriflunomide ≥3 months were included in analyzing drug persistence and safety. Among them, 96 patients treated ≥6 months were included in assessing drug effectiveness in aspects of no evidence of disease activity (NEDA) 3. The number and total volume of PRL were calculated in 76 patients with baseline susceptibility-weighted imaging (SWI), and their association with NEDA3 failure during teriflunomide treatment was investigated., Results: Over a treatment period of 19.7 (3.1-51.7) months, teriflunomide reduced annualized relapse rate (ARR) from 1.1 ± 0.8 to 0.3 ± 0.5, and Expanded Disability Status Scale (EDSS) scores remained stable. At month 24, the NEDA3% and drug persistence rate were 43.8% and 65.1%, respectively. In patients with a baseline SWI, 81.6% had at least 1 PRL, and 42.1% had ≥4 PRLs. The total volume of PRL per patient was 0.3 (0.0-11.5) mL, accounting for 2.3% (0.0%-49.0%) of the total T2 lesion volume. Baseline PRL number ≥ 4 (OR = 4.24, p = 0.009), younger onset age (OR = 0.94, p = 0.039), and frequent relapses in initial 2 years of disease (OR = 13.40, p = 0.026) were associated with NEDA3 failure. The PRL number and volume were not reduced ( p = 0.343 and 0.051) after teriflunomide treatment for more than 24 months. No new safety concerns were identified in this study., Conclusion: Teriflunomide is effective in reducing ARR in Chinese patients with RRMS. Patients with less PRL burden, less frequent relapses, and relatively older age are likely to benefit more from teriflunomide, indicating that PRL might be a valuable measurement to inform clinical treatment decision., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tan, Li, Li, He, ZhangBao, Zhou, Yang, Zhao, Lu, Dong, Li and Quan.)

Continuous exposure to airborne pesticides causes their gradual accumulation in the human body, eventually posing a threat to human health. To the best of our knowledge, risk assessment study of pesticide non-occupational exposure to residents in agricultural areas has not been conducted in China. In this study, air samples (gas and dust) were collected from inside and outside residences of seven households and an area near the field in a grain-growing area (wheat and maize rotation) for eight months, and the pesticides present were examined both qualitatively and quantitatively. Using a 95% confidence interval, 9 out of 16 pesticides were detected, namely acetamiprid, acetochlor, atrazine, flucarbazone-sodium, imidacloprid, methyldisulfuron-methyl, nicosulfuron-methyl, pendimethalin, and beta-cyhalothrin, and their safety was subsequently evaluated. The results showed that the inhalation exposure of households to beta-cyhalothrin exceeded the acceptable range in the first residential, and the excess lifetime cancer risk of acetochlor inhalation exposure in six households and area around the field exceeds 1E-6, which highlights the need to strengthen preventive screening for cancer risk., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction and Objective: The ConcePTION project aims to improve the way medication use during pregnancy is studied. This includes exploring the possibility of developing a distributed data processing and analysis infrastructure using a common data model that could form a foundational platform for future surveillance and research. A prerequisite would be that data from various data access providers (DAPs) can be harmonised according to an agreed set of standard rules concerning the structure and content of the data. To do so, a reference framework of core data elements (CDEs) recommended for primary data studies on drug safety during pregnancy was previously developed. The aim of this study was to assess the ability of several public and private DAPs using different primary data sources focusing on multiple sclerosis, as a pilot, to map their respective data variables and definitions with the CDE recommendations framework., Methods: Four pregnancy registries (Gilenya, Novartis; Aubagio, Sanofi; the Organization of Teratology Information Specialists [OTIS]; Aubagio, Sanofi; the Dutch Pregnancy Drug Register, Lareb), two enhanced pharmacovigilance programmes (Gilenya PRIM, Novartis; MAPLE-MS, Merck Healthcare KGaA) and four Teratology Information Services (UK TIS, Jerusalem TIS, Zerifin TIS, Swiss TIS) participated in the study. The ConcePTION primary data source CDE includes 51 items covering administrative functions, the description of pregnancy, maternal medical history, maternal illnesses arising in pregnancy, delivery details, and pregnancy and infant outcomes. For each variable in the CDE, the DAPs identified whether their variables were: identical to the one mentioned in the CDE; derived; similar but with a divergent definition; or not available., Results: The majority of the DAP data variables were either directly taken (85%, n = 305/357, range 73-94% between DAPs) or derived by combining different variables (12%, n = 42/357, range 0-24% between DAPs) to conform to the CDE variables and definitions. For very few of the DAP variables, alignment with the CDE items was not possible, either because of divergent definitions (1%, n = 3/357, range 0-2% between DAPs) or because the variables were not available (2%, n = 7/357, range 0-4% between DAPs)., Conclusions: Data access providers participating in this study presented a very high proportion of variables matching the CDE items, indicating that alignment of definitions and harmonisation of data analysis by different stakeholders to accelerate and strengthen pregnancy pharmacovigilance safety data analyses could be feasible., (© 2023. The Author(s).)

The pesticide acetochlor (ACT) is a chiral isomer commonly detected in the global environment, yet its specific impacts on liver function remain poorly understood. We utilized zebrafish and L02 cells as research models to comprehensively investigate how ACT and its chiral isomers affect the liver. Our investigations unveiled that the R, Rac, and S isomers of ACT disrupt hepatic lipid transport, catabolism, and synthesis, leading to delayed yolk sac absorption and the accumulation of lipids in zebrafish embryos. These isomers induce oxidative stress in the liver of zebrafish embryos, reducing antioxidant levels and enzyme activity. The accumulated lipids in the liver render it susceptible to oxidative stress, further exacerbating hepatocyte damage. Hepatocyte damage manifests as extensive vacuolization of liver cells and alterations in liver morphology, which are induced by R, Rac, and S. Furthermore, we elucidated the molecular mechanisms underpinning the disturbance of hepatic lipid metabolism by R, Rac, and S in L02 cells. These compounds stimulate lipid synthesis through the upregulation of the AMPK/SREBP-1c/FAS pathway while inhibiting lipolysis via downregulation of the PPAR-α/CPT-1a pathway. Remarkably, our results highlight that S exhibits significantly higher hepatotoxicity in comparison to R. This study provides valuable insights into the hepatic effects of ACT chiral isomers., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier B.V.)

Excessive sulfite usage in food and pharmaceutical production causes respiratory and neurological diseases, underscoring the need for a sensitive and rapid quantification strategy. The portable sensing platform based on a luminescent hydrogel sensor is a powerful tool for the on-site, real-time detection of sulfite ions. However, the lack of recyclability in almost all reaction-based hydrogel sensors increases the application cost. This study constructed a reversible and upconversion nanoprobe combining upconversion nanoparticles (UCNPs) and pararosaniline (PAR) for sulfite detection. The upconversion nanoprobe was further encapsulated in a three-dimensional polyacrylamide hydrogel matrix to create a background-free, reversible hydrogel sensor. The near-infrared excitation of UCNPs avoids the autofluorescence in the hydrogel and real samples. Meanwhile, PAR serves as a specific recognition unit for sulfite ions. After the addition of sulfites, a specific reaction occurs between PAR and sulfites, leading to the recovery of characteristic emission at 540 nm, achieving sensitive detection of sulfite ions. Importantly, this specific reaction is reversible under thermal treatment, allowing the hydrogel sensor to return to its initial state and thus enabling reversible detection of sulfite ions. Furthermore, a portable sensing platform is developed to realize point-of-care, real-time quantitative detection of sulfite ions. The proposed upconversion reversible hydrogel sensor provides a new sensing strategy for the detection of hazardous substances in food and offers new insights into the preparation of reversible, highly sensitive hydrogel sensors.

Excessive acetochlor residues present ecological and food safety challenges. Here, broiler chicks were exposed to varied acetochlor doses to first assess its effects on the gut. Subsequent dietary supplementation with omega-3 was used to assess its anti-contamination effects. Pathologically, acetochlor induced notable ileal lesions including inflammation, barrier disruption, tight junction loss, and cellular anomalies. Mechanistically, acetochlor stimulated the TNFα/TNFR1 and TLR4/NF-κB/NLRP3 pathways, promoting RIPK1/RIPK3 complex formation, MLKL phosphorylation, NLRP3 inflammasome activation, Caspase-1 activation, and GSDMD shearing with inflammatory factor release. These mechanisms elucidate ileal cell death patterns essential for understanding chicken enteritis. Omega-3 supplementation showed promise in mitigating inflammation, though its precise counteractive role remains unclear. Our findings suggest early omega-3 intervention offered protective benefits against acetochlor's adverse intestinal effects, emphasizing its potential poultry health management role. Harnessing dietary interventions' therapeutic potential will be pivotal in ensuring sustainable poultry production and food safety despite persistent environmental contaminants., 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 © 2023. Published by Elsevier Inc.)

Phagocytosis "offense" is a crucial process to protect the organism from diseases and the effects of foreign particles. Insects rely on the innate immune system and thus any hindrance to phagocytosis may greatly affect their resistance to diseases and response to pathogens. The European honeybee, a valuable species due to its economic and environmental contribution, is being challenged by colony collapse disorder leading to its decline. Exposure to multiple factors including pesticides like imidacloprid and amitraz may negatively alter their immune response and ultimately make them more susceptible to diseases. In this study, we compare the effect of different concentrations and mixtures of imidacloprid and amitraz with different concentrations of the immune stimulant, zymosan A. Results show that imidacloprid and amitraz have a synergistic negative effect on phagocytosis. The lowered phagocytosis induces significantly higher hemocyte viability suggesting a negatively correlated protective mechanism "defense" from pesticide-associated damage but may not be protective from pathogens., Competing Interests: Declaration of competing interest The authors declare that there research was conducted in the absence of any conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Given the serious threat of acetochlor (ACT) to the aquatic ecological environment, designing wastewater treatment-oriented adsorbents for the sustainable remediation of actual ACT-contaminated water is a promising yet challenging strategy. Herein, a novel P-doped biochar (PBC-800) with a high adsorption capacity (51.34 mg g -1 ) and a rapid reaction rate (47.35 mg g -1 h -1 ) for ACT was prepared through pyrolyzing of rice straw biomass pre-impregnated with potassium dihydrogen phosphate (KH 2 PO 4 ). Additionally, P-doped biochars synthesized at different pyrolysis temperatures exhibited significant variations in ACT adsorption performance, which was mainly ascribed to the distinction between hydrophilicity and sp 2 conjugate C (I D /I G  = 0.84-1.08). The adsorption behavior of ACT on PBC-800 followed the Elovich kinetics and Freundlich adsorption isotherm models. Thermodynamic calculations indicated that the adsorption of ACT by PBC-800 was a spontaneously disordered decreasing exothermic process. Besides, PBC-800 exhibited a powerful anti-interference for ACT adsorption within complex water matrices, highlighting its potential for various of practical applications. Through characterization analysis and further experiments, it was proved that the excellent adsorption performance of PBC-800 on ACT was ascribed to a combination of physical and chemical adsorption mechanisms, including 57.5% pore filling, 23.4% hydrophobic interaction, 12.7% π-π interaction, and 6.4% hydrogen bonding. Moreover, PBC-800 exerted a prominent adhesion impact upon Gram-positive and negative bacteria at 3 h. This study offers a new idea for the utilization of agricultural residues and provides insights into the mechanism of ACT adsorption through its derived biochar., 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 © 2023 Elsevier Ltd. All rights reserved.)

Aim: Comparative effectiveness research using real-world data often involves pairwise propensity score matching to adjust for confounding bias. We show that corresponding treatment effect estimates may have limited external validity, and propose two visualization tools to clarify the target estimand. Materials & methods: We conduct a simulation study to demonstrate, with bivariate ellipses and joy plots, that differences in covariate distributions across treatment groups may affect the external validity of treatment effect estimates. We showcase how these visualization tools can facilitate the interpretation of target estimands in a case study comparing the effectiveness of teriflunomide (TERI), dimethyl fumarate (DMF) and natalizumab (NAT) on manual dexterity in patients with multiple sclerosis. Results: In the simulation study, estimates of the treatment effect greatly differed depending on the target population. For example, when comparing treatment B with C, the estimated treatment effect (and respective standard error) varied from -0.27 (0.03) to -0.37 (0.04) in the type of patients initially receiving treatment B and C, respectively. Visualization of the matched samples revealed that covariate distributions vary for each comparison and cannot be used to target one common treatment effect for the three treatment comparisons. In the case study, the bivariate distribution of age and disease duration varied across the population of patients receiving TERI, DMF or NAT. Although results suggest that DMF and NAT improve manual dexterity at 1 year compared with TERI, the effectiveness of DMF versus NAT differs depending on which target estimand is used. Conclusion: Visualization tools may help to clarify the target population in comparative effectiveness studies and resolve ambiguity about the interpretation of estimated treatment effects.

The effect of the structure of promising antioxidant agents with prospective medical use, i.e., unsubstituted and para -substituted annelated triazinylacetic acid hydrazides, on their melting points, thermal stabilities, pyrolysis and oxidative decomposition stages and the type of volatiles emitted under heating with the use of DSC and TG/DTG/FTIR/QMS methods was evaluated and discussed. The melting point of the investigated compounds increased with an enhanced number of electrons (directly correlated with their molecular weight). Melting enthalpy values were determined and presented for all the studied compounds. The pyrolysis and oxidative decomposition processes of the analysed molecules consisted of several poorly separated stages, which indicated a multi-step course of the decomposition reactions. It was found that the thermal stability of the tested compounds depended on the type of substituent at the para position of the phenyl moiety or its absence. In both atmospheres used (air and helium), the thermal stability increased in relation to R as follows: -CH 3 ≤ -OCH 3 < -H < -OC 2 H 5 . In an inert atmosphere, it was higher by approx. 8-18 °C than in an oxidative atmosphere. The pyrolysis was connected with the emission of NH 3 , HCN, HNCO, HCONH 2 , HCHO, CO 2 , CO and H 2 O in the case of all the tested compounds, regardless of the substituent attached. In the case of the derivative containing the para -CH 3 group, para -toluidine was an additional emitted aromatic product. In turn, emissions of aniline and alcohol (methanol or ethanol) for compounds with the para -OCH 3 and para -OC 2 H 5 groups, respectively, were confirmed. In oxidative conditions, the release of NH 3 , NO, HCN, HNCO, HCONH 2 , CO 2 , H 2 O and cyanogen (for all the compounds) and para -toluidine (for the para -CH 3 derivative), aniline (for para -OCH 3 , para -OC 2 H 5 and unsubstituted derivatives) and acetaldehyde (for the para -OC 2 H 5 derivative) were clearly observed. No alcohol emissions were recorded for either compound containing the para -OCH 3 - or para -OC 2 H 5 -substitututed phenyl ring. These results confirmed that the pyrolysis and oxidative decomposition of the investigated annelated triazinylacetohydrazides occurred according to the radical mechanism. Moreover, in the presence of oxygen, the reactions of volatiles and residues with oxygen (oxidation) and the combustion process additionally proceeded.

Rhipicephalus (Boophilus) microplus, an invasive species to Africa, and the endemic R. (B.) decoloratus are of high economic importance in the cattle industry. Invasion of the alien species in South Africa has mostly been reported for traditional communal grazing areas where it seemed to be rapid and, in some cases, even replaced the native species. The alien species is also assumed to already be resistant to acaricides upon invasion. The presence of R. (B.) microplus on commercial farms was therefore investigated and resistance screening of both species to field concentrations of cypermethrin, amitraz, and chlorfenvinphos was determined by means of the larval immersion test. Results showed that only 3.7% (of 383) tick collections submitted were R. (B.) microplus populations. A further 1.6% (of 383) showed co-existence of the two species. Comparing the level of resistance to the acaricides between the two species indicated a mean phenotypic resistance of 66.2 and 26.5% of R. (B.) decoloratus populations to cypermethrin and amitraz, respectively. This was significantly lower for R. (B.) microplus, with 23.0 and 4.1% of its populations resistant to cypermethrin and amitraz, respectively. Closed commercial farming areas seemed to have a preventative advantage for the invasion of R. (B.) microplus and displacement of R. (B.) decoloratus by R. (B.) microplus. Regular monitoring of these two species may be of high importance to prevent unnecessary financial losses due to insufficient control and increased awareness of the threat of Asiatic babesiosis vectored by R. (B.) microplus., (© 2023. The Author(s).)

Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet- o -toluidine (AAOT) and o -toluidine (OTD), and adductome analysis was performed. Principal component analysis-discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.

Objective: The degree of substitution (DS) of HES describes the average proportion of substituted glucose molecules in a starch molecule. Although no quantitative studies of the in vivo behavior of the DS have been conducted so far, most pharmacokinetic studies to date have measured HES concentrations using the enzymatic method. This method assumes that at any point in time after an infusion, the DS in a serum remains constant and is identical to the DS in the infused solution. In the present study, we examined the changes in the DS of HES 130/0.42 in vivo in an animal model and compared two methods of measuring HES concentrations in plasma (the enzymatic and the o-Toluidine method)., Methodology: We randomized 22 pigs into 2 groups. After induction of anesthesia, the pigs received 500 ml or 1000 ml of HES 130/0.42 (Tetraspan®). The DS was measured directly after the infusion, then after 30, 60, 120 and 240 minutes. In determining the DS, the hydroxyethyl starch was extracted from the plasma and hydrolyzed with hydrochloric acid to form non-substituted glucose and hydroxyethyl glucose. Subsequently, the concentration of free unsubstituted glucose was determined enzymatically and the total concentration of all (i.e., substituted and unsubstituted) glucose molecules was determined using the o-Toluidine method. From this, the concentration of the substituted glucose (hydroxyethyl glucose) and the DS could be calculated. In addition, the HES concentration was measured first in vitro and then in vivo at any point after the infusion by both the enzymatic method and the o-Toluidine method., Results: The DS increased significantly directly after the infusion from 0.42 to 0.53 (for 500ml) or to 0.50 (for 1000ml); 4 hours later this had further increased to 0.55 and 0.54, respectively (p <0.0001). The HES concentration in vitro showed no significant difference (p = 0.17) when determined with the enzymatic and the o-Toluidine method. In contrast, the serum concentrations in vivo displayed significant differences (p<0.0001) between the two measurement methods. Immediately after the infusion of 500ml HES, the concentration measured with the o-Toluidine method was 31% higher than the one measured with the enzymatic method; 4 hours later, this discrepancy was still at 25%. For 1000 ml HES, the differences amounted to 16% and 25%, respectively., Conclusion: The DS of HES in vivo increases significantly over time. As a result, an HES concentration measured with the enzymatic method in vivo will be significantly lower than the same concentration determined with the o-Toluidine method. In future pharmacokinetic studies, HES concentrations should be measured using a method that takes into account changes in the DS in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lukasewitz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Leflunomide is an immunosuppressive drug indicated for the treatment of rheumatoid arthritis (RA). While the pharmacokinetics (PK) of its active metabolite A771726 reportedly show large interindividual variability, no efficient dose individualization strategy is currently available. The goal of this work was to develop a population PK model for A771726 and propose an optimal individualized dosing strategy., Methods: A771726 plasma concentration data were collected from 50 healthy male volunteers participating in two leflunomide PK studies given a single oral dose of 40 mg. Concentrations were elevated in low body weight (WT) subjects and showed multiple peaks. Thus, A771726 PK modeling was conducted incorporating allometry scaling and enterohepatic circulation (EHC). For dose optimization, simulating a set of 1000 virtual subjects from the developed model and dividing the subjects into 5 groups with WT of 50, 60, 70, 80, 90 kg, respectively, the optimal dose was explored that achieves the drug concentration most similar to the target, which was defined as the concentration for the 70 kg subject treated with the current standard dosage regimen (the loading dose of 100 mg QD for 3 days, followed by the maintenance dose of 20 mg QD)., Results: The data were best described by a two compartment model with first order absorption incorporating EHC with the bile released into the intestine. None of the covariates tested was found to be significant other than WT used in allometry. Simulation showed that the optimal loading dose increased by 15 mg for every 10 kg increment in WT while the optimal maintenance dose was 15 and 25 mg for 50 and 90 kg groups, respectively, and the same (= 20 mg) for the others. Large concentration differences from the target observed in low and high WT groups disappeared when optimal doses were given., Conclusions: This work demonstrates the importance of a population PK model-based dose optimization approach in maintaining drug therapeutic concentrations in leflunomide treatment., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest, (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the recent years control of potentially genotoxic impurities have an increasing importance in the analysis of active pharmaceutical ingredients. Guidelines of different regulatory bodies specify very low limits for these impurities, in many cases the analytical development is challenging to comply with the requirements. In this paper potential genotoxic impurities of Crotamiton drug substance are investigated, a simple and robust gas chromatographic method is developed for the determination of genotoxic impurities in Crotamiton drug substance. One of the main benefits of this method is that it can quantify all potential genotoxic impurities that can be present in the entire synthesis pathway of Crotamiton drug substance. Crotamiton is synthesized from toluidine derivatives, in which the aromatic amine group represents genotoxic alert. In the method development toluidine isomers (o-, m-, p-toluidine), N-ethyl-toluidine isomers (o-, m-, N-ethyl-p-toluidine) and N-methyl-toluidine isomers (o-, m-, N-methyl-p-toluidine) were separated, and the developed gas chromatographic method is validated in accordance with the relevant guidelines at a specification limit of NMT 40 ppm., 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 © 2021 Elsevier B.V. All rights reserved.)

Background: Amitraz is a broad-spectrum insecticide/acaricide for the control of aphids, psyllids, ticks and mites. Current evidence suggests that ticks and phytophagous mites have developed strong resistance to amitraz. Previous studies have shown that multiple mechanisms are associated with amitraz resistance in ticks, but very few reports have involved Panonychus citri. We therefore used whole genome sequencing and bulked segregant analysis (BSA) to identify the mechanism underlying P. citri's resistance to amitraz., Results: High-quality assembly of the whole P. citri genome was completed, resulting in a genome of approximately 83.97 Mb and a contig N50 of approximately 1.81 Mb. Gene structure predictions revealed 11 577 genes, of which 10 940 genes were annotated. Trait-associated regions in the genome were mapped with bulked segregant analysis and 38 candidate SNPs were obtained, of which T752C had the strongest correlation with the resistant trait, located at the 5' untranslated region (UTR) of the β-2R adrenergic-like octopamine receptor gene. The mutation resulted in the formation of a short hairpin loop structure in mRNA and gene expression was down-regulated by more than 50% in the amitraz-resistant strain. Validation of the T752C mutation in field populations of P. citri found that the correlation between the resistance ratio and the base mutation was 94.40%., Conclusion: Our results suggest that this 5' UTR mutation of the β-2R octopamine receptor gene, confers amitraz resistance in P. citri. This discovery provides a new explanation for the mechanism of pest resistance: base mutations in the 5' untranslated region of target gene may regulate the susceptibility of pests to pesticides., (© 2021 Society of Chemical Industry.)

Panonychus citri (McGregor) is the most common pest in citrus-producing regions. Special low-toxicity acaricides, such as spirocyclic tetronic acids and mite growth inhibitors, have been used for a long time in China. However, pesticide resistance in mites is a growing problem due to the lack of new acaricide development. Wide-spectrum insecticides, such as amitraz have gained acceptance among fruit growers. An amitraz-resistant strain of P. citri was obtained by indoor screening to examine field resistance monitoring of mites to acaricides and to explore the resistant mechanism of mites against amitraz. The amitraz-resistant strain of P. citri had an LC50 value of 2361.45 mg l-1. The resistance ratio was 81.35 times higher in the resistant strain of P. citri compared with the sensitive strain. Crossing experiments between the sensitive and resistant strains of P. citri were conducted, resulting in a D value of 0.11 for F1 SS♀×RS♂ and 0.06 for F1 RS♀×SS♂. Reciprocal cross experiments showed that the dose-mortality curves for the F1 generations coincided, indicating that the resistance trait was not affected by cytoplasmic inheritance. The dose-expected response relationship was evaluated in the backcross generation and a significant difference was observed compared with the actual value. The above results indicate that the inheritance of resistance trait was incompletely dominant, governed by polygenes on the chromosome. Synergism studies demonstrated that cytochrome P450s and esterase may play important roles in the detoxification of amitraz. Based on differential gene analysis, 23 metabolism-related genes of P. citri were identified, consistent with the results of synergism studies. Real-time PCR verification implied that P450s, ABC transporters, and acetylcholinesterase might influence the detoxification of amitraz by P. citri. These results provide the genetic and molecular foundation for the management of pest mite resistance.

The impact of increasing resistance of mosquitoes to conventional pesticides has led to investigate various unique tools and pest control strategies. Herein, we assessed the potency of flupyradifurone, a novel pesticide, on fourth instar larvae of Culex quinquefasciatus Say. Further, we evaluated the synergistic action of piperonyl butoxide (PBO) and the octopamine receptor agonists (OR agonists) chlordimeform (CDM) and amitraz (AMZ) on the toxicity of flupyradifurone in comparison with sulfoxaflor and nitenpyram to increase their toxicity on Cx. quinquefasciatus. Results demonstrated that flupyradifurone was the most potent pesticide followed by sulfoxaflor and nitenpyram. Further, the synergetic effect of PBO, CDM, and AMZ was significant for all selected pesticides especially flupyradifurone. However, AMZ had the most significant effect in combination with the selected pesticides followed by CDM and PBO. The toxicity of the pesticides was time-dependent and increased over time from 24, 48, to 72 h of exposure in all experiments. The results indicate that flupyradifurone is a promising component in future mosquito control programs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Varroa destructor is one of the greatest threats for the European honeybee, Apis mellifera. Acaricides are required to control mite infestation. Three conventional chemical acaricide substances are used in France: tau-fluvalinate, flumethrin and amitraz. Tau-fluvalinate was used for over 10 years before experiencing a loss of effectiveness. In 1995, bioassay trials showed the first mite resistance to tau-fluvalinate. In some countries, amitraz was widely used, also leading to resistance of V. destructor to amitraz. In France, some efficiency field tests showed a loss of treatment effectiveness with amitraz. We adapted the bioassay from Maggi and collaborators to determine mite susceptibility to tau-fluvalinate and amitraz in France in 2018 and 2019. The lethal concentration (LC) which kills 90% of susceptible mite strains (LC 90 ) is 0.4 and 12 µg/mL for amitraz and tau-fluvalinate, respectively. These concentrations were chosen as the determining factors to evaluate mite susceptibility. Some mites, collected from different apiaries, present resistance to amitraz and tau-fluvalinate (71% of the mite samples show resistance to amitraz and 57% to tau-fluvalinate). As there are few active substances available in France, and if mite resistance to acaricides continues to increase, the effectiveness of the treatments will decrease and therefore more treatments per year will be necessary. To prevent this situation, a new strategy needs to be put in place to include mite resistance management. We suggest that a bioassay would be a good tool with which to advise the policymakers.

The parasitic mite Varroa destructor is a major pest of the western honeybee, Apis mellifera. The development of acaricide resistance in Varroa populations is a global issue. Discriminating concentrations of acaricides are widely used to detect pest resistance. Two methods, using either glass vials or paraffin capsules, are used to screen for Varroa resistance to various acaricides. We found the glass vial method to be useless for testing Varroa resistance to acaridices, so we developed a polypropylene vial bioassay. This method was tested on tau-fluvalinate-, acrinathrin-, and amitraz-resistant mite populations from three apiaries in Czechia. Acetone was used as a control and technical grade acaricide compounds diluted in acetone were applied to the polypropylene vials. The solutions were spread on the vial surface by rolling the vial, and were then evaporated. Freshly collected Varroa females were placed in the vials and the mortality of the exposed mites was measured after 24 h. The Varroa populations differed in mortality between the apiaries and the tested compounds. Mites from the Kyvalka site were resistant to acrinathrin, tau-fluvalinate, and amitraz, while mites from the Postrizin site were susceptible to all three acaricides. In Prelovice apiary, the mites were susceptible to acrinathrin and amitraz, but not to tau-fluvalinate. The calculated discriminating concentrations for tau-fluvalinate, acrinathrin, and amitraz were 0.66, 0.26 and 0.19 µg/mL, respectively. These results indicate that polyproplyne vial tests can be used to determine discriminating concentrations for the early detection of acaricide resistant Varroa. Finally, multiple-resistance in Kyvalka may indicate metabolic resistance.

The evolution of tick resistance to acaricides in New Caledonia was followed in two ways: through two large scale surveys in 1998 and 2014 and through the routine analysis of samples of ticks with suspected resistance. The results of the two approaches were qualitatively similar though analysis of ticks with suspected resistance gave higher frequencies of resistance, as expected of a biased sample. Resistance tests using a larval packet test have been conducted since 1993 for deltamethrin and 2003 for amitraz. Deltamethrin was used in country-wide control from 1986 to 2003 and amitraz since 1996. This study analyzed the variation of resistance parameters such as lethal concentration 50 (LC50) and the resistance factor over 21 years for deltamethrin and LC99 over 11 years for amitraz. There was an increase in deltamethrin's annual mean LC50 from 1993 to 2004 when it reached 2.9 g/l and then a progressive decrease, reaching a level consistently below 1g/l since 2007. Even though breeders stopped using deltamethrin in New Caledonia in 2003, the percentage of susceptible strains has remained below 30% since 1998. Amitraz's LC99 mean increased significantly from 0.31 g/l in 2003 to 2.96 g/l in 2014. Whereas all tested strains in 2003 were susceptible, only 40% of strains tested at the request of farmers were susceptible in 2014. The recent territory-wide survey showed that 76.7% of strains are still susceptible. This study established that resistance to amitraz has developed slowly in New Caledonia. A reversion phenomenon may have occurred concerning deltamethrin resistance, visible through the decrease of LC50 mean and the decreased proportion of very resistant strains; however the proportion of susceptible strains remains at a low level and there is anecdotal evidence that high resistance can re-emerge rapidly., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Field tick isolates of Rhipicephalus (Boophilus) microplus were collected from eleven districts located in the northern and eastern states of India to access the resistance status to "Amitraz". Adult immersion test was optimized using laboratory reared acaricide susceptible IVRI-I line and minimum effective concentration was determined as 487.7 ppm with 95 % confidence interval of 455.8-521.8. The discriminating concentration was determined as 975.4 ppm and was tested on female ticks collected by two stage stratified sampling from organized dairy farms and villages. Based on three variables, viz.,mortality, egg masses and reproductive index, the resistance level was categorized.Resistance to amitraz was detected at level I in 3 isolates (RF = 1.56-5.0), at level II in 6 isolates (RF = 9.3-23.3) and at level III in 1 isolate (RF = 27.3) whereas one isolate was found susceptible. The highest resistance was found in the SKR isolate (RF = 27.3) and minimal resistance was detected in the N-24P isolate (RF = 1.56). These experimental data will help in designing tick control strategy which is suffering from acaricide failure and to overcome development of resistance in ticks.

Formamidines are unique insecticides and acaricides that elicit multiple effects in controlling insects. Here, we tested two formamidines, amitraz, and chlordimeform, for their synergistic actions on type II pyrethroids and neonicotinoids to increase their larvicidal actions on the fourth instars of Aedes aegypti L. An organophosphate insecticide was used as a negative control. After 24 h, the synergism of formamidines was highest on imidacloprid, followed by two type II pyrethroids, deltamethrin and fenvalerate. After 48 h, the synergism of formamidines on imidacloprid decreased, remained unchanged on type II pyrethroids, and increased noticeably on two of the newer type neonicotinoids, dinotefuran and thiamethoxam. By 72 h, synergism of formamidines on dinotefuran reached the maximum, while that on imidacloprid was at a minimum. Both formamidines did not show synergistic effects on permethrin or fenitrothion. In all cases, the synergistic effects of amitraz on the two major classes of larvicides were greater than for chlordimeform. These results indicate that amitraz is a promising synergist that shows the potential to increase the efficacy of certain members of type II pyrethroids as well as neonicotinoids to control Ae. aegypti larvae.

The objectives of this study were: (1) to determine the prevalence and factors associated with Rhipicephalus microplus , resistant to cypermethrin and amitraz, from cattle farms in Veracruz, Mexico, and (2) to determine in vitro mortality percentages of field populations of R. microplus exposed to discriminating doses (DD) of cypermethrin and amitraz. Fifty-three populations of R. microplus were tested by bioassays using DD of cypermethrin (0.05%) and amitraz (0.0002%). The prevalence of cattle farms with R. microplus ticks that were resistant to cypermethrin and amitraz, and co-resistant to both acaricides, was 90.6, 54.7, and 47.2%, respectively. The level of cypermethrin resistance, measured as a survival percentage, was higher as compared to amitraz. Cattle farms with ≤50 animals (odds ratio [OR]  =  3.84, 95% confidence interval [CI]  =  1.07-13.70, P  =  0.038) and a stocking density of >1 animal unit per ha (AU/ha) (OR  =  4.21, 95% CI  =  1.0-17.71, P  =  0.050) had a higher probability to develop R. microplus tick populations co-resistant to both acaricides. In conclusion, there is a high prevalence of R. microplus populations on cattle farms in Veracruz, Mexico that are both resistant to cypermethrin and amitraz and co-resistant to both acaricides. The level of cypermethrin resistance is critical, and the exposition variables of ≤50 cattle and a stocking density of >1 AU/ha were factors associated with R. microplus co-resistant to both acaricides.

Amitraz, a formamidine acaricide, has been reported to act as a synergist of contact insecticides and acaricides in both agriculture and animal health. A laboratory contact test was therefore conducted to determine whether amitraz at 12.5 ppm could improve the contact potency of fipronil on ticks. A controlled glass vial bioassay was used to assess the efficacy of fipronil alone, amitraz alone, and fipronil plus amitraz on unfed adult Rhipicephalus sanguineus. Assessments of lethality were made at 6, 24, and 48 h after the introduction of the ticks to the vials. No significant mortality was observed in the control treatment or in the amitraz alone treatment. Concentration and time dependent mortality rates were observed in ticks exposed to fipronil alone or fipronil plus amitraz, with higher mortality observed in the latter group. Results from this study gave synergistic EC50 ratios between fipronil alone and fipronil plus amitraz of >7.3, 137 and 97 at 6, 24, and 48 h, respectively. A similar response was seen at the EC90 level. These results indicate that fipronil was synergized by amitraz in this adult tick residual contact study. The addition of amitraz to fipronil also provided a significant improvement in the speed of kill.

Commercial formulation of the amitraz is the most used acaricide in the southern region of Rio Grande do Sul State, however scientific data about the efficacy of these products in this region are sparse. The aim of the present study was to evaluate the frequency of R. (B.) microplus populations resistant to amitraz acaricides in the Southern region of Rio Grande do Sul State. So, the efficacies of five commercial products containing amitraz were compared. Results showed that the efficacy of amitraz acaricides was lower than 69% in 23% of studied populations. Nearly 29% of the tick samples showed an acaricide efficacy equal to zero, suggesting the presence of a high level of resistance to amitraz. Moreover, the most used commercial product in the region showed the lowest efficacy index. These results alert about the possible spreading of resistant on tick populations and justify research directed toward the search for alternative products to control this parasite.

In Venezuela, the acaricides, amitraz and cypermethrin, are widely used to control tick infestations in cattle infested with Boophilus microplus. This work aims to determine the in vitro efficacy of amitraz and cypermethrin through the adult immersion test. A sample of 480 engorged females was collected from four dairy farms (180 ticks/farm) and divided in groups of 10 ticks averaging 220 mg/tick. Commercial preparation of amitraz and cypermethrin were used to prepare the working concentration of amitraz (52, 104, 208, 416 and 832 ppm), and cypermethrin (37.5, 75, 150, 300, 600 ppm). The control groups were submerged only in water. Ticks were placed in a glass lamina at 27 degrees C and 85% relative humidity (RH) and incubated for 18 days. The eggs masses were collected, weighed, separated in vials, and allowed to hatch at 85% RH and 27 degrees C for 21 days. Both the emerged larvae and eggs were quantified in each tube in order to determine the efficacy percentage and the resistance factor. The efficacy average to amitraz was 30.89%, 50.75%, 50.62%, 59.72%, and 60.53% at concentrations of 52, 104, 208, 416, and 832 ppm, respectively. The efficacy of cypermethrin was 14.3%, 11.82%, 17.25%, 20.23%, and 43.23% at concentrations of 37.5, 75, 150, 300, and 600 ppm, respectively. The amitraz resistance factor to each farm was 3.14, 1.19, 1.88, and 1.89, whereas with cypermethrin it was 8.89, 6.03, 7.12, and 18.45. Statistically significant differences between the effectiveness of different concentrations of amitraz or cypermethrin were not observed. Higher concentrations do not increase the efficacy of the products.

The aim of this work was to develop a technique of acaricides evaluation by immersion of larvae. Larvae were obtained from 30 engorged females of Riphicephalus (Boophilus) microplus were collected in a ranch in Lages, Santa Catarina state, Brazil. Larvae were obtained from 10mg of eggs placed into 5ml disposable syringes, previously prepared and kept under controlled conditions of laboratory, and collected between the third and the twelfth day of oviposition. Seven to ten days old larvae were submitted to the acaricide laboratory test, in ten replications, by their immersion in eight dilutions of commercial acaricides (amitraz or cypermethrin), for 30 seconds; for each replication and commercial product, a control group was immersed in distilled water. Results were evaluated 24 hours after each treatment by counting the number of live and dead larvae. The mean LD50 values and standard error of the mean for cypermethrin and amitraz were 2.99 +/- 0.36 and 17.22 +/- 4.20 ppm, respectively, for a confidence interval of 95%. The proposed acaricide laboratory test procedures by larvae immersion offered promising results for the evaluation of acaricides.

A novel spot-on formulation containing metaflumizone plus amitraz (ProMeris/ProMeris Duo for Dogs, Fort Dodge Animal Health, Overland Park, KS) was evaluated in four laboratory studies to confirm efficacy against fleas and ticks on dogs for 1 month. Three different strains of cat flea (Ctenocephalides felis felis) and four tick species were used. Rhipicephalus sanguineus and Dermacentor variabilis were evaluated concurrently in two studies and Ixodes scapularis and Amblyomma americanum in one study each. In all studies, dogs were randomly allocated to treatment groups and compared with nontreated dogs. One study also included a placebo treatment and a commercial product containing fipronil plus S-methoprene. All treatments were applied to the skin at a single spot between the scapulae on Day 0. Dogs were infested with fleas and/or ticks prior to treatment and then reinfested at weekly intervals for 6 weeks after treatment and evaluated for efficacy at 1 or 2 days after treatment and each reinfestation. These studies confirmed that treatment with ProMeris for Dogs at the proposed commercial dose rate rapidly controlled existing infestations of fleas and ticks on dogs. Treatment provided control of reinfesting fleas for up to 6 weeks and at least 4 weeks control of ticks. Efficacy was confirmed in a variety of dog breeds against three different flea strains and four common species of ticks found on dogs in the United States.

The control of the southern cattle tick, Boophilus microplus (Canestrini), in Mexico and many other countries relies on chemical acaricides. Boophilus microplus has developed resistance to all major classes of acaricides in recent years. To gain a better understanding of the resistance and to develop resistance management strategies that benefit both Mexican ranchers and USDA's cattle fever tick eradication program (CFTEP), the authors used larval bioassay techniques to determine levels of resistance to permethrin and amitraz and then evaluated synergism between these two acaricides in one susceptible laboratory tick strain and four resistant strains originating from Mexico and Brazil. To examine mechanisms of resistance to permethrin in these strains, the frequency of a mutated sodium channel gene was determined using a PCR assay. The tick strains from Mexico and Brazil demonstrated 49.4- to over 672.2-fold resistance to permethrin, and up to 94.5-fold resistance to amitraz. While the San Roman strain from Mexico was the most permethrin-resistant strain, the Santa Luiza strain from Brazil was the most amitraz-resistant strain. A significant correlation was found between the permethrin resistance ratio and the allelic frequency of the sodium channel mutation. Significant synergism between permethrin and amitraz was found when one acaricide was tested in the presence of another. Synergism ratios ranged from 1.5 to 54.9 when amitraz was tested as a synergist for permethrin. Similar synergism ratios were obtained when permethrin was tested as a synergist for amitraz. Permethrin caused virtually no mortality in the San Roman strain, even at the highest concentration (3294 microg cm(-2)). Adding amitraz (11.0 microg cm(-2)) to permethrin led to a dramatic increase in larval mortality, even at very low concentrations of permethrin., (2007 Society of Chemical Industry)

The ability of the Miller, Soberanes, and White bioassay techniques to describe amitraz susceptibility in three different strains of Boophilus microplus (Canestrini) (Acari: Ixodidae) was compared. For a susceptible strain, all techniques adequately described amitraz susceptibility by producing a full range of mortality that corresponded with increasing concentration of amitraz. However, when resistant strains were evaluated, only the Miller and the Soberanes techniques adequately estimated the dose-response relationship. Lethal concentrations were not precisely estimated when all the data were included in the analyses for every strain and technique tested. Better estimates were obtained when subsets of data around the range of interest were subjected to probit analysis. For the Soberanes technique, the slope of the probit regression was steeper for the Brazilian resistant and Texan susceptible strains compared with the heterozygous Mexican strain. The pattern was different when the same strains were tested with the Miller technique. The slopes of the regressions for the Mexican and the Texan strains did not differ significantly, but the Brazilian strain had a steeper slope than the other strains. Resistance ratios were much greater when the Soberanes technique was used than when the Miller technique was used on the same strains. However, neither technique produced enough separation between susceptible and resistant strains to develop a traditional discriminating dose (DD) test that required a concentration of 2x LC99.9 estimate. A DD test at the LC99 would be possible for both techniques. We discuss the strengths and weaknesses of the three techniques, including potential improvements to the White technique. The White technique has the greatest potential to determine the mechanisms of amitraz resistance in detailed synergist studies. Currently, only the Miller method can fulfill this task. The Miller and Soberanes techniques are well suited for the study of the epidemiology of resistance worldwide, because they use commercially available, formulated amitraz that is easy and inexpensive to obtain.

Laboratory trials were realized between the year 2001 at 2004 to evaluate the efficacy of commercial ixodicides against samples of Boophilus microplus collected of dairy bovines in 17 properties of eight cities at Paraíba Valey region, State of São Paulo Brazil. The trials was performed against engorged female using immersion technique. It was evidenced low efficacy of products of the chemical groups synthetic pyrethroid (28.24%) and amitraz (47.19%). The best efficacy was presented by products of the association between synthetic pyrethroid with organophosphorus (88.64%).

The tick Boophilus microplus represents a serious pathological constraint to livestock production in New Caledonia. Cattle ticks are controlled by chemical application of two acaricides that are currently used in New Caledonia; deltamethrin is used at 46% of the cattle production facilities and amitraz at the remaining 54% premises where resistance to deltamethrin has been identified. In 2003, a modified Larval Packet Test (LPT) was used to conduct a survey for amitraz resistance. Ticks were collected from 29 farms, including farms using deltamethrin (n=8) or amitraz (n=21). Of eighteen different tick populations, sixteen populations were defined susceptible to amitraz and two populations were considered amitraz-resistant. This is the first report of populations of B. microplus being resistant to amitraz, using the modified LPT in New Caledonia. A thorough survey of tick susceptibility to amitraz in cattle farms of the country should be conducted to assess the presence of amitraz-resistant populations. The emergence of amitraz resistance so soon after its introduction has some important implications for the strategy and organisation of tick control in New Caledonia, and this paper discusses some of the urgent actions that should be undertaken.

A series of in vitro and in vivo bioassays were conducted to assess the ectoparasiticide activity of isopropyl-4-nitro-2,6-bis(trifluoromethyl)-1-benzimidazole-carbamate, an experimental benzimidazole-carbamate class compound. This compound was less potent than permethrin against ectoparasiticide-susceptible larvae of the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae); larvae of the southern cattle tick, Boophilus microplus (Canestrini); and adult stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) in vitro, but it was significantly more potent than permethrin against the Santa Luiza strain of B. microplus known to possess high-level resistance to amitraz and pyrethroids. In contrast, the benzimidazole-carbamate was substantially more efficacious than permethrin when applied topically onto rats that were infested with A. americanum nymphs. These results suggest that this experimental compound may be a viable candidate ectoparasiticide that retains significant activity against resistant B. microplus and also suggests that the benzimidazole-carbamate chemistry may be useful for addressing the growing problem of resistance in ectoparasites.

Brimer et al. (Vet. Parasitol. 51: 123-135, 1993 and 59: 249-255, 1995) developed a migration assay for acaricidal effect of acetylcholinesterase inhibitors and macrocyclic lactones utilising Sarcoptes scabiei var. suis mites. In contrast to many others, this assay is fully quantitative but quite time-consuming. The aim of the present investigation was to modify this assay to become faster and simpler. As a result accurate determinations can now be obtained within 6h, as opposed to 24h. Furthermore it was demonstrated that also Otodectes cynotis mites can be used with only minor modifications of the procedures. The cholinesterase inhibitor diazinon and the formamide amitraz were used as acaricides. Thus, the mite migration assay now has been proven useful for acaricidal compounds belonging to three chemical groups with different modes of action, namely organophosphorous cholinesterase inhibitors, macrocyclic lactones acting on the glutamanergic/GABAegic motoneurons, and formamide inhibitors of the octopamine systems of arthropods.

The method is based on the enhancement effect of the resonance light-scattering (RLS) of pararosaniline by DNA. In the pH rang of 0.5-1.5, the enhanced intensity of pararosaniline RLS at 355 nm is proportional to the concentration of DNA in the rang of 0.20-15 micrograms.mL-1. The detection limit is 36 ng.mL-1. This method is simple, rapid and has been applied satisfactorily to the determination of DNA in mixed samples.

Deer self-treatment devices ('4-posters') were evaluated for their efficacy in reducing populations of blacklegged ticks, Ixodes scapularis, and lone star ticks, Amblyomma americanum. At each of three locations in Maryland, 25 '4-posters' were operated in study areas of approximately 5.18 km2. Populations of host-seeking ticks were monitored by flagging of treated areas and similar untreated control areas without '4-posters.' From 1998 to 2002 the percent mortalities achieved were 69, 75.8 and 80 at the three study sites infested with I. scapularis nymphs, and 99.5 and 95.3 for A. americanum nymphs at the two sites where this species occurred.

White-tailed deer treated themselves with a commercial pour-on acaricide formulation containing 2% amitraz as they fed from an ARS-patented '4-poster' topical treatment device. Whole kernel corn attracted deer to a single device placed in each of two deer-fenced pastures. In the treatment pasture, the rollers of the treatment device were charged with the acaricide, whereas the rollers of the device in the other pasture remained untreated. Deer were allowed to use the '4-posters' during periods of tick activity beginning in early to midspring and lasting through late summer to early fall for three consecutive years. Pretreatment sampling of adults and nymphs with dry-ice traps and larval masses with flip cloths showed no significant differences in population indices between the two pastures; however, after the third year of treatment, control of nymphal and adult ticks in the treated pasture was 91.9 and 93.7%, respectively, when compared with the untreated pasture. Control of larval masses increased from 68.4% in year 1 to 96.4% in year 2, but declined to 88.0% in year 3, probably because of the presence of feral hogs. This study demonstrated that application of amitraz to white-tailed deer through free-choice interaction with a '4-poster' device significantly reduced the abundance of free-living lone star ticks in a deer-fenced experimental pasture. Moreover, the yearly pattern of incremental increases in control and the final percentage control values for all three parasitic life stages in this topical application study were similar in magnitude to that observed in a previously conducted study in which the systemic acaricide ivermectin was used to reduce populations of free-living ticks by controlling ticks on deer.

Integral immunohistochemical analysis of immune responses in frozen sections requires that, in addition to constitutively expressed membrane CD markers, less stable determinants can be reliably visualized. Therefore, we compared the commonly used acetone fixation method with pararosaniline fixation for six determinant categories. These categories included selected constitutively expressed markers, inducible co-stimulatory molecules, pro- and anti-inflammatory cytokines (including the novel cytokine IL-18, also known as IGIF and IL-1gamma), antigen-specific antibody in plasma cells, bacterial peptidoglycan, and lysosomal acid phosphatase activity. Human spleen and mouse spleen activated by agonistic anti-CD40 antibody or TNP-Ficoll immunization were analyzed in parallel with brain tissue from multiple sclerosis (MS) patients and marmoset monkeys with experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Fixation with pararosaniline resulted in better morphology of all tissues and inhibited endogenous alkaline phosphatase activity in brain tissue. Most determinants could be reliably detected. Staining sensitivity and intensity were markedly increased for selected determinant-tissue combinations, e.g., for IL-4 in human spleen and CD40 in human and mouse spleen. These data show that pararosaniline is a useful alternative to acetone, resulting in superior morphology and specific staining for selected determinant-tissue combinations. This provides additional flexibility for in situ analysis of immune reactivity.

Objective: This study assessed the clinical performance of dentin-bonded crowns, in which ceramic crowns are bonded to underlying dentin with a resin composite-based luting material and a dentin bonding agent., Method and Materials: Twenty-five patients who had received such restorations more than 1 year previously were recalled for evaluation of their crowns., Results: Sixty dentin-bonded complete-coverage restorations were assessed. Forty-one of the crowns had been placed on incisor teeth. The mean time since placement of the restorations was 2.43 years. Fifty-seven of the 60 restorations were intact. The three failures had resulted from cracks in the restorations, which had not clinically debonded. No secondary caries was detected at the crown margins, and anatomic form was assessed as excellent for 56 crowns. Root canal treatment had been required in one case. Color match was rated very good for 47 crowns. All 25 patients were satisfied with their restorations., Conclusion: Dentin-bonded crowns may be found to have a low rate of failure and to provide a high level of patient satisfaction.