Assessment of the molecular mechanisms of action of novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one allosteric modulators at the M1 muscarinic acetylcholine receptors

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer's disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism (Mistry et al., 2016), but their molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, BQCA, in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist, ACh, on three different signalling pathways, (ERK1/2 phosphorylation, IP1 accumulation and β-arrestin-2 recruitment), revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated

Biochemical and molecular mechanisms of acaricide resistance in Tetranychus urticae

The two-spotted spider mite Tetranychus urticae is an economically important pest in many agricultural crops worldwide. Its high reproductive potential and extremely short life cycle, combined with the frequent acaricide applications usually required to maintain the population below economic threshold, facilitates rapid resistance build-up. This has led to the development of resistance against almost all commercially used compounds. In this study we tried to unravel the mechanisms behind the resistance against a number of acaricides with different mode of action at the toxicological, biochemical, genetic and molecular level. In a first series of experiments, we investigated pyrethroid resistance in Greek T. urticae strains. Combined data from toxicological bioassays and biochemical and synergistic studies indicated that although enhanced P450 mono-oxygenase activities were associated with resistance, target site insensitivity proved to be the major resistance component. In order to get a better insight in the latter, a 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III, which still has an unknown role. Also, three alternative splicing variants were identified, of which two correspond to the mutually exclusive k/l exon pair already identified in insects. The mode of inheritance of resistance was confirmed to be almost completely recessive, which is consistent with studies on the target site mechanism for pyrethroids in other organisms. In T. urticae, organophosphate and carbamate resistance is often caused by acetylcholinesterase insensitivity. By combining toxicological, biochemical and molecular data from three reference laboratory and three organophosphate resistant (OP) strains, the AChE1 mutations associated with resistance in T. urticae were characterised. The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. Compared to the reference strains, the insecticide concentration needed to inhibit 50% of the AChE1 activity in the OP strains was at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the carbamate carbaryl. By comparing the AChE1 sequence between laboratory and OP strains, four amino acid substitutions were detected: (1) F331W which was present in all three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. F331W, G328A and A201S are possibly involved in resistance to organophosphate and carbamate insecticides. These findings were also confirmed by means of a structural model. F331W is probably the most important and the most common in T. urticae. It can be easily detected by a diagnostic PCR-RFLP assay developed in this study. We also evaluated the possible antagonism of organophosphate and carbamate insecticides on the toxicity of bifenazate in T. urticae when applied in mixtures. Bifenazate, a new and frequently used carbazate, is a pro-acaricide which needs to be activated by carboxylesterases. Two OP strains were used and several organophosphate (chlorpyrifos, azinphos methyl and phosmet) and carbamate (carbaryl and methomyl) insecticides were evaluated. Mixing chlorpyrifos with bifenazate strongly decreased bifenazate toxicity in both tested strains. However, in the strain with a higher esterase activity, antagonism decreased after 2 days. Of all other tested chemicals, only methomyl displayed an antagonistic effect 1 day after treatment. These findings indicate that mixing organophosphate and carbamate insecticides with bifenazate may inhibit bifenazate efficacy under field conditions, especially when resistant strains are present. Spirodiclofen, a recently developed and commercialized acaricide, is a selective, non-systemic tetronic acid derivative. In order to develop strategies to minimise resistance in the field to spirodiclofen, we selected a T. urticae population for spirodiclofen resistance in the laboratory. This selection yielded a strain with a resistance ratio of 274, determined on the larval stage. This strain was used to determine genetic, toxicological, biochemical and cross-resistance data. The egg stage of the resistant strain remained far more susceptible than the mobile stages. No cross-resistance was found against other acaricides, except for spiromesifen, another tetronic acid derivative. Based on synergist experiments and enzyme assays, it appeared that especially P450 mono-oxygenases, but also esterases and glutathione-S-transferases, could be involved in the metabolic detoxification of spirodiclofen. Pre-treatment of the resistant females with the synergists PBO or DEF could increase the inhibitory effect of spirodiclofen on reproduction, again demonstrating the possible involvement of mono-oxygenases and esterases. Among the esterases of T. urticae, probably only a few are more expressed in spirodiclofen resistant strains, and these apparently prefer the purpose-designed substrate 1-naphthyl 2,2-dimethylbutyrate. This was also confirmed by separating esterase isozymes by native isoelectric focusing. Because spirodiclofen interferes with lipid biosynthesis, total lipid content was measured in female adults. No significant differences between treated and non-treated female adults were found, both in the susceptible and resistant strain. However, the total lipid content in the resistant females was significantly higher than in susceptible females. The genetic analysis using crossing experiments showed that the resistance is inherited as an intermediate trait under control of more than one gene. Resistance to spirodiclofen in the laboratory selected strain exceeded by far the recommended field rate, showing the capacity for quick resistance development in the field. A good acaricide resistance management programme is necessary to prevent fast resistance build-up in the field. However, without selection pressure, resistance tends to be unstable and can decrease in the presence of susceptible individuals owing to the intermediate, polygenic inheritance mode. No evidence was found to support the existence of other resistance mechanisms, like mutations in the target site (ACCase) or overproduction of the target enzyme. In order to facilitate designing new ACCase inhibitor insecticides and understanding mechanism of resistance to currently used ACCase inhibitors, we tried to clone and express T. urticae ACCase using different expression systems. The most promising results were achieved by a baculovirus expression system and also by heat-shocking Codon Rare E. coli cells with the pET-ACC vector. More efforts should be done to overcome several problems in expressing this enzyme e.g. by optimizing conditions to get higher levels of expression, protein solubility, activity and stability. We also performed a practical screening in Dutch cut rose glasshouses due the fact that growers complained on failing spider mite control. In order to check whether control failure was caused by resistance, the susceptibility of 15 strains, sampled from infested roses, was tested to 11 acaricides: tebufenpyrad, milbemectin, abamectin, cyflumetofen, bifenthrin, spiromesifen, hexythiazox, etoxazole, bifenazate, acequinocyl and chlorpyrifos. Three doses were used to discern between resistance and susceptibility: the recommended field dose (FD), FD/5 and FD x 5. Ten of these strains were screened for known resistance mechanism such as elevated detoxifying enzyme activities and previously reported resistance mutations using biochemical and molecular diagnostics. Potential cross-resistance between acaricides was estimated by correlation analysis. The strains showed different levels of resistance to tested acaricides. Twelve out of 15 strains showed resistance against hexythiazox, and 9 out of 15 against bifenthrin and tebufenpyrad. Two strains were found which showed high levels of resistance to most tested insecticides, even against one which has not been registered in Europe. A high correlation was found between some acaricides, e.g. between abamectin and milbemectin. Without selection pressure, resistance seemed to be unstable in most cases, after one year. All field strains had an increased metabolic activity, compared to the susceptible strains. A number of amino acid substitutions known to be involved in resistance to different groups were present in different strains. The best example was the presence of the F331W substitution in the acetylcholine esterase, which was present in all field strains tested. The results obtained in this research may all together lead to a better understanding of acaricide resistance in T. urticae, which, in turn, may lead to more efficient resistance management programs

Species diversity of soil mites (Acari: Mesostigmata) under different agricultural land use types

Mites are among the most important members of soil arthropod communities, because they are the most diverse in terms of ecological niche and behavior. Due to the sensitivity of soil mites to soil disturbance, their diversity and numbers can be used as ecological indices for assessing disturbances in ecosystems. To determine the effect of land use type on soil mite biodiversity, abundance and biodiversity indices of soil inhabiting mesostigmatic mites were evaluated at eight sites in Saman and Shahrekord, Iran, each site including two adjacent agricultural pieces of land: an orchard and a crop field. The biodiversity of mites was measured by several biodiversity indices and then compared by analysis of variance. The specimens collected belonged to 12 families, 17 genera and 24 species. The biodiversity index values calculated in different months showed that these indices were usually higher in warm months and in orchards than in cold months and crop fields. In the examined crop fields, the diversity index values were lower after harvesting, probably due to soil disturbance by agricultural machinery. There was a significant difference in the Shannon-Wiener's diversity index among different land uses. The maximum and minimum values of this index were recorded at the vine orchard (1.48) and wheat field (0.15) in an elm/wheat site at Shahrekord, respectively. The soil organic matter content was maximum in the vineyard (2.12%) and minimum in the wheat field (0.41%)

Metabolic mechanisms of resistance to spirodiclofen and spiromesifen in Iranian populations of Panonychus ulmi

The European red mite, Panonychus ulmi (Koch), is one of the major pests of apple trees worldwide. Cyclic ketoenol compounds such as spimdiclofen and spiromesifen are frequently used to control phytophagous spider mites in agricultural crops, including P. ulmi on apple trees. Spider mites, however, can rapidly develop resistance against acaricides and, in this study, multiple P. ulmi populations from apple orchards in Iran were monitored for spimdiclofen and spiromesifen resistance. The Urmia and Shahin Dej population showed the highest spirodiclofen resistance ratio (more than 150-fold) compared to the susceptible Ahar population. Toxicity bioassays also revealed the presence of moderate cross-resistance between spiromesifen and spirodiclofen, but not towards the chitin synthase inhibitor etoxazole. As a first step towards elucidating spirodiclofen resistance mechanisms, the role of detoxification enzymes (cytochmme P450 monooxygenases, carboxyl/choline esterases and glutathione 5-transferases) was investigated by in vivo synergism and in vitro enzyme assays. PBO pretreatment synergized spimdiclofen toxicity in the populations of Urmia and Shahin Dej to a higher extent than in the susceptible Ahar population. Furthermore, enzyme activity measurements showed relatively higher activity of detoxifying enzymes in the resistant populations. In conclusion, increased detoxification is most likely underlying spimdiclofen resistance and results in limited cross-resistance to spimmesifen

Characterization of abamectin resistance in Iranian populations of European red mite, Panonychus ulmi Koch (Acari: Tetranychidae)

The European red mite, Panonychus ulmi Koch is one of the most important pests in apple orchards and was introduced to Iran by apple seedlings from Europe. The insecticide/acaricide abamectin for example has been used extensively against P. ulmi and some other pests in apple orchards. To evaluate abamectin resistance in field-collected populations of P. ulmi, 12 populations were collected from commercial apple orchards of East Azarbaijan, West Azarbaijan, and Isfahan provinces. The abamectin toxicity was determined by a leaf disc spray method. The LC(50)( )values of abamectin ranged from 0.11 mg a. iL(-1) to 5.50 mg a. i.L-1. All field populations were resistant to abamectin (RR ranged from 11- to 46-fold) in comparing with PSR-TK, a reference susceptible population for abamectin. The Mahabad population was identified as the most resistant population. The glutamate-gated chloride channels (GluCls) are well-known target site of abamectin in mites and it was demonstrated that amino acid substitutions in GluCls can confer abamectin resistance. The partial channels PuGluC11, PuGluC12, and PuGluC13 were sequenced in Mahabad population, but the previously reported point mutations associated with abamectin resistance in Tetranychus urticae Koch were not found. In contrast, the cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) significantly increased abamectin toxicity in Mahabad (synergistic ratio SR= 64), a moderately resistant population, and Shahin Dej (SR = 2) population. Also, pretreatment of triphenylphosphate (TPP) resulted in reduced LC50 values of abamectin in Shahin Dej (SR = 4.79) and Mahabad (SR = 8.91) populations. The second highest synergism ratio (SR = 22.13) against abamectin was observed in the resistant population of Mahabad with the glutathione S-transferase inhibitor diethylmaleate (DEM). Although quantification of activity of detoxification enzymes with model substrates did not support the role of detoxification enzymes, the synergism assays and the lack of target-site resistance suggested that multiple metabolic mechanisms are involved in abamectin resistance

First report of Altenia mersinella (Lep.: Gelechiidae: Gelechiinae: Litini), a pest of pistachio, from Iran

During a survey on pistachio, Pistacia vera L., pests in Isfahan, Iran, in 2015, some lepidopteran larvae living and feeding inside galls of the wrinkling pistachio aphid, Forda hirsuta Mordv., were observed. After laboratory rearing of these larvae, the moths were identified as Altenia mersinella (Staudinger, 1879) (Lep.: Gelechiidae) by the third author. This species has previously been reported from Turkey, Cyprus, Syria, Lebanon and Palestine but it is the first report of this species from Iran. Based on the available literature, this is also the first record of a host plant for this species

Point mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Iranian populations of the European red mite Panonychus ulmi

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maracieh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F15381, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field

Molecular and genetic analysis of resistance to METI-I acaricides in Iranian populations of the citrus red mite Panonychus citri

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found

Prediction of length of stay for stroke patients using artificial neural networks

Strokes are neurological events that affect a certain area of the brain. Since brain controls fundamental body activities, brain cell deterioration and dead can lead to serious disabilities and poor life quality. This makes strokes the leading cause of disabilities and mortality worldwide. Patients that suffer strokes are hospitalized in order to be submitted to surgery and receive recovery therapies. Thus, it’s important to predict the length of stay for these patients, since it can be costly to them and their family, as well as to the medical institutions. The aim of this study is to make a prediction on the number of days of patients’ hospital stays based on information available about the neurological event that happened, the patient’s health status and surgery details. A neural network was put to test with three attribute subsets with different sizes. The best result was obtained with the subset with fewer features obtaining a RMSE and a MAE of 5.9451 and 4.6354, respectively.FCT - Fundação para a Ciência e a Tecnologia (UID/CEC/00319/2019