Phytotoxic phenols from the needles of Cedrus deodara.

During the course of screening for anti-seed germination phytochemicals, the methanol fraction of the Cedrus deodara fresh needles showed potent activity. Bioactivity-guided fractionation led to the isolation of thirty-eight phenolic compounds. Four ones were identified as previously undescribed including (7S,8S)-3-methoxy-9'-acetoxy-3',7-epoxy-8,4'-oxyneoligna-4,9-diol (7), (7S,8R)-dihydro-3'-hydroxy-8-acetoxymethyl-7-(4-hydroxy-3-methoxy-phenyl)-1'-benzofuranpropanol (10), (8S)-4,9,9'-trihydroxy-3,3'-dimethoxy-8,4'-oxyneolignan (11) and (7S,8S)-4,7,9'-trihydroxy-3,3'-dimethoxy-9-acetoxy-8,4'-oxyneolignan (16), respectively. The potential phytotoxic effects of these compounds on the seed germination and root elongation of Arabidopsis thaliana were evaluated by the filter paper assay developed in our laboratory. Bioassay results indicated that caffeic acid (36) displayed most significant inhibitory activities against the seed germination and root elongation of A. thaliana, stronger than those of the commercial herbicides acetochlor and glyphosate at the same concentration of 200 µg/mL. Ditetrahydrofuran lignan (1), dihydrochalcone (25), and eight simple phenols (28, 29, 31, 33-35, 37 and 38) completely inhibited the seed germination of A. thaliana at the concentration of 400 µg/mL, which were as active as acetochlor. Dihydroflavone (21) and the simple phenols 32-34 displayed stronger inhibitory effects on the root elongation of A. thaliana than that of glyphosate. The inhibitory effects of these active compounds on the seed germination and root elongation of Amaranthus tricolor and Lactuca sativa were evaluated as well. The phytotoxic activity of 11, 16, 22, 25, 31, 34, 37 and 38 were detected for the first time. In addition, the structure-activity relationships of the same class of these phytochemicals were discussed.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

Target identification and acaricidal activity difference of amitraz and its metabolite DPMF in Tetranychus cinnabarinus (Boisduval).

BACKGROUND: Amitraz is a broad-spectrum formamidine acaricide proven to be effective against mites in all development stages. Under acidic conditions, amitraz is hydrolyzed to N2 -(2,4-dimethylphenyl)-N1 -methyformamidine (DPMF), an active metabolite for mite control. Octopamine and tyramine receptors are well known targets of amitraz. Until now, no research has been conducted about the amitraz target in Tetranychus cinnabarinus. This study aimed to identify the target genes of amitraz in T. cinnabarinus and reveal the mechanisms behind the differential acaricidal activities of amitraz and DPMF. RESULTS: Analysis of the toxicity, stress expression, target sensitivity and binding site of amitraz against T. cinnabarinus showed that TcOctß2R was the main target gene of amitraz. DPMF had more potent acaricidal activity against T. cinnabarinus and was more effective at activating TcOctß2R than amitraz. Furthermore, the three synergists had no significant effect on amitraz and DPMF, indicating that the detoxification metabolism was not related to the difference in acaricidal activity. CONCLUSION: In this study, TcOctß2R was identified as the main target gene of amitraz against T. cinnabarinus. The divergence of target binding was responsible for the difference in acaricidal activity between amitraz and DPMF. The results also revealed the physiological and pharmacological functions of octopamine receptors (OARs) in T. cinnabarinus and could provide a basis for the design of new acaricides, with OARs as a special target. © 2023 Society of Chemical Industry.

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress.

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.

Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNFα-induced inflammatory responses.

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 µM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.

Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.

Human dihydroorotate dehydrogenase (hDHODH), as the fourth and rate-limiting enzyme of the de novo pyrimidine synthesis pathway, is regarded as an attractive target for malignancy therapy. In the present study, a novel series of teriflunomide derivatives were designed, synthesized, and evaluated as hDHODH inhibitors. 13t was the optimal compound with promising enzymatic activity (IC50 = 16.0 nM), potent antiproliferative activity against human lymphoma Raji cells (IC50 = 7.7 nM), and excellent aqueous solubility (20.1 mg/mL). Mechanistically, 13t directly inhibited hDHODH and induced cell cycle S-phase arrest in Raji cells. The acute toxicity assay indicated a favorable safety profile of 13t. Notably, 13t displayed significant tumor growth inhibition activity with a tumor growth inhibition (TGI) rate of 81.4% at 30 mg/kg in a Raji xenograft model. Together, 13t is a promising inhibitor of hDHODH and a preclinical candidate for antitumor therapy, especially for lymphoma.

Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination.

BACKGROUND AND OBJECTIVES: To test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models. METHODS: The effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting-sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein-GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin. RESULTS: In cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination. DISCUSSION: TF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.

Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Tick treatment practices in the field: Access to, knowledge about, and on-farm use of acaricides in Laikipia, Kenya.

The prevention of tick-borne diseases is a major challenge for livestock production globally. Tick control strategies include the use of acaricides, but the prescribed strategies do not achieve the desired results in several countries, including Kenya. To better understand how tick treatment practices, contribute to reported tick treatment failures, we assessed livestock owners' acaricide procurement, level of knowledge about acaricides and tick resistance, and how they apply acaricides. We also assessed the quality of the commonly available acaricides. We focused on three livestock systems in Laikipia County, Kenya: two private ranches; one community ranch whose members communally graze their cattle and acquire and apply acaricides; and individual livestock owners in two pastoral communities who individually graze their cattle and acquire and apply acaricides. Through interviews and focus group discussions we assessed; access to acaricides, livestock owners' knowledge, and acaricide use practices; interview data were triangulated with participant observations (n = 107). We analysed nine commonly used acaricides to determine the active ingredient concentration and we determined the concentration of active ingredients in acaricide dilutions collected on farms. All livestock owners had access to and used chemical acaricides for tick control, predominantly amitraz-based. Private ranchers bought one amitraz-based acaricide in bulk directly from the manufacturer, while all other livestock owners bought from agrovet shops. The livestock owners acquired knowledge about acaricides from their own experiences and through experience-based recommendations from peers, but not from the technical information provided by the manufacturers and agrovet shops. All pastoral livestock frequently changed acaricide brand and active ingredient class. A large majority of pastoralists (86%) mixed acaricide brands within and across active ingredient classes; a smaller majority (56%) mixed acaricides with crop pesticides and insecticides. Our lab tests confirmed the content description on the labels bought from agrovet shops. However, on-farm acaricide dilutions from all three livestock systems deviated from the level recommended for effective treatment. If too diluted, the acaricide does not kill ticks, promoting resistance development. If too concentrated, this increases environmental contamination and raises public health concerns. Livestock owners lack a technical understanding of the functioning of acaricides, compromising their use and effectiveness. The widely adopted mixing of acaricides with insecticides and pesticides raises serious health concerns.

Mechanisms of amitraz resistance in a Rhipicephalus microplus strain from southern Brazil.

Amitraz is one of the most used acaricides for the control of ticks of domestic animals, however, extensive use of this active ingredient has favored the development of resistant populations of Rhipicephalus microplus worldwide. The possible mechanisms of metabolic and/or target-site alterations mechanisms of amitraz resistance were investigated in a Brazilian field population of R. microplus (São Gabriel strain). Bioassays with the synergists piperonylbutoxide, triphenylphosphate and diethyl-maleate were used to evaluate the metabolic mechanisms involved. Target-site insensitivity was investigated by amplification and sequencing of a fragment of the octopamine/tyramine (OCT/TYR) receptor gene. Piperonylbutoxide synergism (synergism ratio = 2.8) indicated the participation of the P450 pathway in the detoxification of amitraz. Previously reported single nucleotide polymorphisms that confer amino acid changes in the OCT/TYR receptor, threonine to proline (T8P) and leucine to serine (L22S), were found in the amitraz-resistant strain but not in the susceptible reference strain. The results suggest that amitraz resistance in the studied strain is multi-factorial and may result from cytochrome P450 detoxification and mutations in octopamine receptors.

Efficacy and safety of dihydroorotate dehydrogenase (DHODH) inhibitors "leflunomide" and "teriflunomide" in Covid-19: A narrative review.

Dihydroorotate dehydrogenase (DHODH) is rate-limiting enzyme in biosynthesis of pyrimidone which catalyzes the oxidation of dihydro-orotate to orotate. Orotate is utilized in the biosynthesis of uridine-monophosphate. DHODH inhibitors have shown promise as antiviral agent against Cytomegalovirus, Ebola, Influenza, Epstein Barr and Picornavirus. Anti-SARS-CoV-2 action of DHODH inhibitors are also coming up. In this review, we have reviewed the safety and efficacy of approved DHODH inhibitors (leflunomide and teriflunomide) against COVID-19. In target-centered in silico studies, leflunomide showed favorable binding to active site of MPro and spike: ACE2 interface. In artificial-intelligence/machine-learning based studies, leflunomide was among the top 50 ligands targeting spike: ACE2 interaction. Leflunomide is also found to interact with differentially regulated pathways [identified by KEGG (Kyoto Encyclopedia of Genes and Genomes) and reactome pathway analysis of host transcriptome data] in cogena based drug-repurposing studies. Based on GSEA (gene set enrichment analysis), leflunomide was found to target pathways enriched in COVID-19. In vitro, both leflunomide (EC50 41.49 ± 8.8 µmol/L) and teriflunomide (EC50 26 µmol/L) showed SARS-CoV-2 inhibition. In clinical studies, leflunomide showed significant benefit in terms of decreasing the duration of viral shredding, duration of hospital stay and severity of infection. However, no advantage was seen while combining leflunomide and IFN alpha-2a among patients with prolonged post symptomatic viral shredding. Common adverse effects of leflunomide were hyperlipidemia, leucopenia, neutropenia and liver-function alteration. Leflunomide/teriflunomide may serve as an agent of importance to achieve faster virological clearance in COVID-19, however, findings needs to be validated in bigger sized placebo controlled studies.

New bioassay cage methodology for in vitro studies on Varroa destructor and Apis mellifera.

Varroa destructor Anderson and Trueman, is an ectoparasitic mite of honey bees, Apis mellifera L., that has been considered a major cause of colony losses. Synthetic miticides have been developed and registered to manage this ectoparasite, however, resistance to registered pyrethroid and organophosphate Varroacides have already been reported in Canada. To test toxicity of miticides, current contact-based bioassay methods are designed to evaluate mites and bees separately, however, these methods are unlikely to give an accurate depiction of how miticides interact at the colony level. Therefore, the objective of this study was to develop a bioassay cage for testing the toxicity of miticides on honey bees and Varroa mites simultaneously using amitraz as a reference chemical. A 800 mL polypropylene plastic cage holding 100-150 bees was designed and officially named "Apiarium". A comparison of the effects of three subsequent dilutions of amitraz was conducted on: Varroa mites placed in glass vials, honey bees in glass Mason jars, and Varroa-infested bees in Apiariums. Our results indicated cumulative Varroa mortality was dose-dependent in the Apiarium after 4 h and 24 h assessments. Apiarium and glass vial treatments at 24 h also had high mite mortality and a positive polynomial regression between Varroa mortality and amitraz dose rates. Moreover, chemical application in the Apiarium was less toxic for bees compared to the Mason jar method. Considering these results, the Apiarium bioassay provides a simple, cheap and reliable method for simultaneous chemical screening on V. destructor and A. mellifera. Furthermore, as mites and bees are tested together, the Apiarium simulates a colony-like environment that provides a necessary bridge between laboratory bioassay testing and full field experimentation. The versatility of the Apiarium allows researchers to test a multitude of different honey bee bioassay experiments including miticide screening, delivery methods for chemical products, or development of new mite resistance-testing methodology.

Effect of Teriflunomide on Cells From Patients With Human T-cell Lymphotropic Virus Type 1-Associated Neurologic Disease.

OBJECTIVE: To test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHODS: PBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 tax and HTLV-1 hbz messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression. RESULTS: In culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 µM (38.3% inhibition), 50 µM (65.8% inhibition), and 100 µM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8+ and CD4+ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or tax mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4+ T cells obtained from a subset of patients with HAM/TSP. CONCLUSIONS: These results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.

Ponesimod Compared With Teriflunomide in Patients With Relapsing Multiple Sclerosis in the Active-Comparator Phase 3 OPTIMUM Study: A Randomized Clinical Trial.

Importance: To our knowledge, the Oral Ponesimod Versus Teriflunomide In Relapsing Multiple Sclerosis (OPTIMUM) trial is the first phase 3 study comparing 2 oral disease-modifying therapies for relapsing multiple sclerosis (RMS). Objective: To compare the efficacy of ponesimod, a selective sphingosine-1-phosphate receptor 1 (S1P1) modulator with teriflunomide, a pyrimidine synthesis inhibitor, approved for the treatment of patients with RMS. Design, Setting, and Participants: This multicenter, double-blind, active-comparator, superiority randomized clinical trial enrolled patients from April 27, 2015, to May 16, 2019, who were aged 18 to 55 years and had been diagnosed with multiple sclerosis per 2010 McDonald criteria, with a relapsing course from the onset, Expanded Disability Status Scale (EDSS) scores of 0 to 5.5, and recent clinical or magnetic resonance imaging disease activity. Interventions: Patients were randomized (1:1) to 20 mg of ponesimod or 14 mg of teriflunomide once daily and the placebo for 108 weeks, with a 14-day gradual up-titration of ponesimod starting at 2 mg to mitigate first-dose cardiac effects of S1P1 modulators and a follow-up period of 30 days. Main Outcomes and Measures: The primary end point was the annualized relapse rate. The secondary end points were the changes in symptom domain of Fatigue Symptom and Impact Questionnaire-Relapsing Multiple Sclerosis (FSIQ-RMS) at week 108, the number of combined unique active lesions per year on magnetic resonance imaging, and time to 12-week and 24-week confirmed disability accumulation. Safety and tolerability were assessed. Exploratory end points included the percentage change in brain volume and no evidence of disease activity (NEDA-3 and NEDA-4) status. Results: For 1133 patients (567 receiving ponesimod and 566 receiving teriflunomide; median [range], 37.0 [18-55] years; 735 women [64.9%]), the relative rate reduction for ponesimod vs teriflunomide in the annualized relapse rate was 30.5% (0.202 vs 0.290; P < .001); the mean difference in FSIQ-RMS, -3.57 (-0.01 vs 3.56; P < .001); the relative risk reduction in combined unique active lesions per year, 56% (1.405 vs 3.164; P < .001); and the reduction in time to 12-week and 24-week confirmed disability accumulation risk estimates, 17% (10.1% vs 12.4%; P = .29) and 16% (8.1% vs 9.9; P = .37), respectively. Brain volume loss at week 108 was lower by 0.34% (-0.91% vs -1.25%; P < .001); the odds ratio for NEDA-3 achievement was 1.70 (25.0% vs 16.4%; P < .001). Incidence of treatment-emergent adverse events (502 of 565 [88.8%] vs 499 of 566 [88.2%]) and serious treatment-emergent adverse events (49 [8.7%] vs 46 [8.1%]) was similar for both groups. Treatment discontinuations because of adverse events was more common in the ponesimod group (49 of 565 [8.7%] vs 34 of 566 [6.0%]). Conclusions and Relevance: In this study, ponesimod was superior to teriflunomide on annualized relapse rate reduction, fatigue, magnetic resonance imaging activity, brain volume loss, and no evidence of disease activity status, but not confirmed disability accumulation. The safety profile was in line with the previous safety observations with ponesimod and the known profile of other S1P receptor modulators. Trial Registration: ClinicalTrials.gov Identifier: NCT02425644.

Characterization and functional analysis of a ß-adrenergic-like octopamine receptor from the oriental armyworm (Mythimna separata Walker).

The ß-adrenergic-like octopamine receptor (OA2B2), which binds the biogenic amine octopamine, belongs to the class of G-protein coupled receptors and significantly regulates many physiological and behavioral processes in insects. In this study, the putative open reading frame sequence of the MsOA2B2 gene in Mythimna separata was cloned, the full-length complementary DNA was 1191 bp and it encoded a 396-amino acid protein (GenBank accession number MN822800). Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. Real-time quantitative polymerase chain reaction of spatial and temporal expression analysis revealed that the MsOAB2 gene was expressed in all developmental stages of M. separata and was most abundant in egg stages and second and fourth instars compared with other developmental stages, while the expression level during the pupal stage was much lower than that at the other stages. Further analysis with sixth instar M. separata larvae showed that the MsOA2B2 gene was expressed 1.81 times higher in the head than in integument and gut tissues. Dietary ingestion of dsMsOA2B2 significantly reduced the messenger RNA level of MsOA2B2 and decreased mortality following amitraz treatment. This study provides both a pharmacological characterization and the gene expression patterns of OA2B2 in M. separata, facilitating further research for insecticides using MsOA2B2 as a target.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

An update on amitraz efficacy against Rhipicephalus microplus after 15 years of disuse.

Amitraz is an acaricide that is widely used in veterinary medicine to control the cattle tick Rhipicephalus microplus. However, controversy exists in the literature regarding the resistance of R. microplus to this product. The present work provides an update on the acaricidal efficacy of amitraz (Triatox®, 12.5 % amitraz) after 15 years without its use on a property. Two in vivo (bovines treated with amitraz and submitted to tick counts, n = 20 animals) and one in vitro (adult immersion test, n = 40 ticks) assays were performed to determine product efficacy. The efficacy of the commercial formulation tested in the first in vivo trial ranged from 14.1 to 47.0%, and in the second from 3.6 to 35.1%, for the 28 days of the experiments. Efficacy for the in vitro trial was 47.38%. The dose recommended by the manufacturer of the product did not cause mortality to most of the ticks of this strain, and efficacy/resistance was not reverted or modified after 15 years (estimated 60 tick generations).

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

Restriction of intracellular Salmonella typhimurium growth by the small-molecule autophagy inducer A77 1726 through the activation of the AMPK-ULK1 axis.

Autophagy plays an important role in restricting the growth of invading intracellular microbes. Salmonella (S) Typhimurium, an intracellular pathogen that causes gastroenteritis and food poisoning in humans, evades autophagic detection by multiple mechanisms. There has been growing interest in developing autophagy inducers as novel antimicrobial agents for treating intracellular bacterial infections. We recently reported that A77 1726, the active metabolite of the anti-inflammatory drug leflunomide, induces autophagy by activating AMP-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1). Our present study aims to determine if A77 1726 was able to restrict intracellular Salmonella growth by inducing autophagy. We first confirmed the ability of A77 1726 to induce autophagy by activating the AMPK-ULK1 axis in uninfected RAW264.7 (a murine macrophage cell line) and HeLa cells (a human cervical carcinoma cell line). A77 1726 enhanced autophagy in S. Typhimurium-infected cells, as evidenced by increased levels of LC3 lipidation and increased numbers of autophagosomes and autolysosomes. Confocal microscopy revealed that A77 1726 induced xenophagy in macrophages, as evidenced by an increased number of LC3-coated bacteria in the cytoplasm. A77 1726 significantly decreased the number of intracellular S. Typhimurium in macrophages. Taken together, our study has demonstrated the ability of A77 1726 to restrict intracellular S. Typhimurium growth in vitro by enhancing xenophagy.

Genotyping amitraz resistance profiles in Rhipicephalus microplus Canestrini (Acari: Ixodidae) ticks from Punjab, India.

Acaricide resistance is one of the greatest threats to sustainable and effective control of vector ticks worldwide. The amitraz resistance status in cattle tick, Rhipicephalus microplus populations collected from 18 districts of Punjab in north-western India were characterized using bioassay and molecular assays. The modified larval packet test was used and the resistance factors (RF) against amitraz for the field populations were in the range of 0.36-4.85, indicating level I resistance status in ten populations. Characterization of a partial segment of the octopamine/tyramine (OCT/Tyr) receptor gene of R. microplus field populations from Punjab revealed a total of 18 nucleotide substitutions in the coding region out of which 5 were non-synonymous substitutions. Three of these non-synonymous substitutions (T8P, V15I and A20 T) were earlier reported in American and South African populations of R. microplus. Among the two single nucleotide polymorphisms (A22C-T8P; T65C-L22S) potentially linked to amitraz resistance in American, South African and Zimbabwean resistant populations, only the T8P substitution was recorded from the Barnala population. The PCR-RFLP assay using EciI restriction enzyme was used for genotyping of the larvae as homozygous resistant (RR), homozygous susceptible (SS) and heterozygous (SR). Genotyping of 514 larval DNA samples from 18 field populations revealed 92.8 % larval population as SR and the remaining 7.2 % as RR genotypes. The percentage of resistant alleles in the tick populations was 53.6 (range 50.0-57.2) indicating its moderate distribution in the region. The present study is the pioneer report establishing the hypothesis that amitraz-resistance is recessively inherited and heterozygous individuals show phenotypic susceptibility to the drug in the Indian tick populations.