Living systematic review and meta-analysis of plasma-concentrations of antipsychotic drugs in carriers and non-carriers of variant CYP450 genotypes: Living systematic review protocol.

Introduction: Carriers of variant alleles of genes that encode liver CYP450 and UGT enzymes may experience abnormal plasma levels of antipsychotics and, consequently, worse efficacy or tolerability. Although pharmacogenomics is a rapidly developing field, current guidelines often rely on limited, underpowered evidence. We have previously demonstrated that meta-analysis is a viable strategy for overcoming this problem. Here, we propose a project that will expand our previous work and create a living systematic review and meta-analysis of drug plasma level differences between carriers and non-carriers of variant genotype-predicted phenotypes for every pharmacokinetic drug-gene interaction relevant to commonly used antipsychotic drugs. Protocol: First, a baseline systematic review and meta-analysis will be conducted by searching for observational pharmacogenomics-pharmacokinetic studies. Data on dose-adjusted drug plasma levels will be extracted, and participants will be grouped based on their genotype for each drug-gene pair separately. Differences in plasma drug levels between different phenotypes will be compared using a random-effect ratio-of-means meta-analysis. The risk of bias will be assessed using ROBINS-I, and the certainty of evidence will be assessed using GRADE. Following the establishment of baseline results, the literature search will be re-run at least once every six months, and the baseline data will be updated and re-evaluated as new evidence is published. A freely available website will be designated to present up-to-date results and conclusions. Discussion: This systematic review will provide evidence-based results that are continuously updated with evidence as it emerges in the rapidly developing field of pharmacogenomics. These results may help psychiatrists in their decision-making, as clinicians are becoming increasingly aware of the patients' genetic data as testing becomes more widespread and cheaper. In addition, the results may serve as a scientific basis for the development of evidence-based pharmacogenomics algorithms for personalized dosing of antipsychotics to mitigate potentially harmful drug-gene interactions.

Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

BACKGROUND: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. OBJECTIVES: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. METHODS: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. RESULTS: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. CONCLUSIONS: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Exploring the Regulation of Cytochrome P450 in SH-SY5Y Cells: Implications for the Onset of Neurodegenerative Diseases.

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, ß-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following ß-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases.

Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

Functional studies of McSTE24, McCYP305a1, and McJHEH, three essential genes act in cantharidin biosynthesis in the blister beetle (Coleoptera: Meloidae).

Cantharidin is a toxic defensive substance secreted by most blister beetles when attacked. It has been used to treat many complex diseases since ancient times and has recently regained popularity as an anticancer agent. However, the detailed mechanism of the cantharidin biosynthesis has not been completely addressed. In this study, we cloned McSTE24 (encoding STE24 endopeptidase) from terpenoid backbone pathway, McCYP305a1 (encoding cytochrome P450, family 305) and McJHEH [encoding subfamily A, polypeptide 1 and juvenile hormone (JH) epoxide hydrolase] associated to JH synthesis/degradation in the blister beetle Mylabris cichorii (Linnaeus, 1758, Coleoptera: Meloidae). Expression pattern analyses across developmental stages in adult males revealed that the expressions of 3 transcripts were closely linked to cantharidin titer exclusively during the peak period of cantharidin synthesis (20-25 days old). In contrast, at other stages, these genes may primarily regulate different biological processes. When RNA interference with double-stranded RNA suppressed the expressions of the 3 genes individually, significant reductions in cantharidin production were observed in males and also in females following McJHEH knockdown, indicating that these 3 genes might primarily contribute to cantharidin biosynthesis in males, but not in females, while females could self-synthesis a small amount of cantharidin. These findings support the previously hypothesized sexual dimorphism in cantharidin biosynthesis during the adult phase. McCYP305a1 collaborates with its upstream gene McSTE24 in cantharidin biosynthesis, while McJHEH independently regulates cantharidin biosynthesis in males.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

A Malpighian Tubule-Specific P450 Gene SlCYP9A75a Contributes to Xenobiotic Tolerance in Spodoptera litura.

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura, especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a, is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

Potential Involvement of MnCYP710A11 in Botrytis cinerea Resistance in Arabidopsis thaliana and Morus notabilis.

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (MnCYP710A11) was discovered in mulberry (Morus notabilis). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea, there was a notable increase in the expression of MnCYP710A11. MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea. The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea, elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H2O2 and O2- levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.

Characterization of the ddt1 Mutant in Rice and Its Impact on Plant Height Reduction and Water Use Efficiency.

Rice (Oryza sativa L.), a fundamental global staple, nourishes over half of the world's population. The identification of the ddt1 mutant in rice through EMS mutagenesis of the indica cultivar Shuhui527 revealed a dwarf phenotype, characterized by reduced plant height, smaller grain size, and decreased grain weight. Detailed phenotypic analysis and map-based cloning pinpointed the mutation to a single-base transversion in the LOC_Os03g04680 gene, encoding a cytochrome P450 enzyme, which results in a premature termination of the protein. Functional complementation tests confirmed LOC_Os03g04680 as the DDT1 gene responsible for the observed phenotype. We further demonstrated that the ddt1 mutation leads to significant alterations in gibberellic acid (GA) metabolism and signal transduction, evidenced by the differential expression of key GA-related genes such as OsGA20OX2, OsGA20OX3, and SLR1. The mutant also displayed enhanced drought tolerance, as indicated by higher survival rates, reduced water loss, and rapid stomatal closure under drought conditions. This increased drought resistance was linked to the mutant's improved antioxidant capacity, with elevated activities of antioxidant enzymes and higher expression levels of related genes. Our findings suggest that DDT1 plays a crucial role in regulating both plant height and drought stress responses. The potential for using gene editing of DDT1 to mitigate the dwarf phenotype while retaining improved drought resistance offers promising avenues for rice improvement.

The Effect of Maternal High-Fat or High-Carbohydrate Diet during Pregnancy and Lactation on Cytochrome P450 2D (CYP2D) in the Liver and Brain of Rat Offspring.

Cytochrome P450 2D (CYP2D) is important in psychopharmacology as it is engaged in the metabolism of drugs, neurosteroids and neurotransmitters. An unbalanced maternal diet during pregnancy and lactation can cause neurodevelopmental abnormalities and increases the offspring's predisposition to neuropsychiatric diseases. The aim of the present study was to evaluate the effect of maternal modified types of diet: a high-fat diet (HFD) and high-carbohydrate diet (HCD) during pregnancy and lactation on CYP2D in the liver and brain of male offspring at 28 (adolescent) or 63 postnatal days (young adult). The CYP2D activity and protein level were measured in the liver microsomes and the levels of mRNAs of CYP2D1, 2D2 and 2D4 were investigated both in the liver and brain. In the liver, both HFD and HCD increased the mRNA levels of all the three investigated CYP2D genes in adolescents, but an opposite effect was observed in young adults. The CYP2D protein level increased in adolescents but not in young adults. In contrast, young adults showed significantly decreased CYP2D activity. Similar effect of HFD on the CYP2D mRNAs was observed in the prefrontal cortex, while the effect of HCD was largely different than in the liver (the CYP2D2 expression was not affected, the CYP2D4 expression was decreased in young adults). In conclusion, modified maternal diets influence the expression of individual CYP2D1, CYP2D2 and CYP2D4 genes in the liver and brain of male offspring, which may affect the metabolism of CYP2D endogenous substrates and drugs and alter susceptibility to brain diseases and pharmacotherapy outcome.

Molecular insights into the functional analysis of P450 CYP321A7 gene in the involvement of detoxification of lambda-cyhalothrin in Spodoptera frugiperda.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a widely recognized global agricultural pest that has significantly reduced crop yields all over the world. S. frugiperda has developed resistance to various insecticides. Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides, leading to increased resistance in insect populations. However, the function of the specific P450 gene for lambda-cyhalothrin resistance in S. frugiperda was unclear. Herein, the expression patterns of 40 P450 genes in the susceptible and lambda-cyhalothrin-resistant populations were analyzed. Among them, CYP321A7 was found to be overexpressed in the resistant population, specifically LRS (resistance ratio = 25.38-fold) derived from a lambda-cyhalothrin-susceptible (SS) population and FLRS (a population caught from a field, resistance ratio = 63.80-fold). Elevated enzyme activity of cytochrome P450 monooxygenases (P450s) was observed for LRS (2.76-fold) and the FLRS (4.88-fold) as compared to SS, while no significant differences were observed in the activities of glutathione S-transferases and esterases. Furthermore, the knockdown of CYP321A7 gene by RNA interference significantly increased the susceptibility to lambda-cyhalothrin. Remarkably, the knockdown of CYP321A7 reduced the enzymatic activity of P450 by 43.7%, 31.9%, and 22.5% in SS, LRS, and FLRS populations, respectively. Interestingly, fourth-instar larvae treated with lambda-cyhalothrin at the LC30 dosage had a greater mortality rate due to RNA interference-induced suppression of CYP321A7 (with increases of 61.1%, 50.0%, and 45.6% for SS, LRS, and FLRS populations, respectively). These findings suggest a link between lambda-cyhalothrin resistance and continual overexpression of CYP321A7 in S. frugiperda larvae, emphasizing the possible importance of CYP321A7 in lambda-cyhalothrin detoxification in S. frugiperda.

Baseline of susceptibility, risk assessment, biochemical mechanism, and fitness cost of resistance to dimpropyridaz, a novel pyridazine pyrazolecarboxamide insecticide, in Bemisia tabaci from China.

Bemisia tabaci is one of the most destructive agricultural insect pests around the world, and it has developed high levels of resistance to most pesticides. Dimpropyridaz, a novel insecticide developed by BASF, displays excellent activity against piercing-sucking insect pests. In this study, baseline of susceptibility showed all tested field populations of B. tabaci are susceptible to dimpropyridaz. After continuous selection with dimpropyridaz in the lab, a B. tabaci strain (F12) developed 2.2-fold higher level of resistance compared with a susceptible MED-S strain, and the realized heritability (h2) was estimated as 0.0518. The F12 strain displayed little cross-resistance to afidopyropen, cyantraniliprole, sulfoxaflor, or abamectin, and significantly increased activity of cytochrome P450 monooxygenase (P450). The fitness cost of dimpropyridaz resistance was evident in F12 strain, which had a relative fitness of 0.95 and significantly lower fecundity per female compared with MED-S strain. Taken together, B. tabaci displays high susceptibility to dimpropyridaz in the field, and low risk of developing resistance to dimpropyridaz under successive selection pressure. Little cross-resistance to popular insecticides was found, and fitness cost associated dimpropyridaz resistance was observed. Higher activity of cytochrome P450 in the F12 strain, may be involved in the process of detoxifying dimpropyridaz in whitefly.

The cytochrome P450 gene CYP4g1 driven by high temperature confers abamectin tolerance on Liriomyza trifolii through promoting cuticular hydrocarbons biosynthesis.

Liriomyza trifolii, an invasive pest, poses a substantial threat to horticultural and vegetable plants. It spreads rapidly, especially in hot weather, leading to large-scale outbreaks with strong thermotolerance and insecticide resistance. In this study, mortality and LtCYP4g1 expression in L. trifolii were evaluated after thermal and insecticides exposure. Furthermore, functional verification of LtCYP4g1 was conducted through RNA interference and bacterial survival assays in Escherichia coli containing recombinant LtCYP4g1 protein. Results indicated that a short time exposure to high temperature incresed insecticide tolerance of L. trifolii, attributed to decreased mortality and induced LtCYP4g1 expression; LtCYP4g1 was involved in stimulating synthesis of cuticular hydrocarbons (CHCs) and elevating epicuticle lipid content and thickness, and E. coli cells overexpressing LtCYP4g1 exhibited significant tolerance to thermal and insecticide stress. In general, P450-mediated tolerance of L. trifolii was enhanced by high temperature, with LtCYP4g1 playing a role in promoting biosynthesis of CHCs for thickening epidermal lipid barrier and reducing cuticular penetration. This study provides a framework for delving into the function of CYP450s in insecticide detoxification and illustrates the role of global warming in driving the evolution of L. trifolii.

Gene-specific transcriptional activation by the Aspergillus fumigatus AtrR factor requires a conserved C-terminal domain.

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. IMPORTANCE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.

Chlorantraniliprole Resistance in Spodoptera frugiperda: Resistance Monitoring, Resistance Risk, and Resistance Mechanisms.

Spodoptera frugiperda is a significant global pest, and chlorantraniliprole (CAP) is extensively used in China for its control. Understanding CAP resistance in S. frugiperda is crucial for effective management of this pest. Field populations exhibited varying degrees of resistance to CAP (RR = 1.74-5.60-fold). After 10 generations of selection, the CAP-resistant strain developed over 10-fold resistance, with a realized heritability (h2) of 0.10. Genetic analysis reveals inheritance patterns as autosomal, incomplete recessive, and monofactorial. The CAP-resistant strain showed limited cross-resistance to lufenuron and tetrachlorantraniliprole, negative cross-resistance to spinetoram, and no observed cross-resistance to other insecticides. Biochemical analysis suggested that P450-mediated detoxification is the primary resistance mechanism, with 26 genes overexpressed in the CAP-resistant strain. Additionally, the knockdown of CYP4L13, CYP6B39, CYP6B40, and CYP4G74 significantly increased the sensitivity of the resistant larvae to CAP. These findings highlight the resistance risk of CAP in S. frugiperda and emphasize the crucial role of P450 enzymes in resistance.

RNA interference of the clock gene period disrupts circadian rhythms in the expression of genes related to insecticide resistance in the chagas disease vector Triatoma infestans (Hemiptera: Reduviidae).

In Triatoma infestans it was observed pyrethroid resistance attributed in part to an elevated oxidative metabolism mediated by cytochromes P450. The nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) plays a crucial role in catalysing the electron transfer from NADPH to all cytochrome P450s. The daily variations in the expression of CPR gene and a P450 gene (CYP4EM7), both associated with insecticide resistance, suggested that their expressions would be under the endogenous clock control. To clarify the involvement of the clock in orchestration of the daily fluctuations in CPR and CYP4M7 genes expression, it was proposed to investigate the effect of silencing the clock gene period (per) by RNA interference (RNAi). The results obtained allowed to establish that the silencing of per gene was influenced by intake schemes used in the interference protocols. The silencing of per gene in T. infestans reduced its expression at all the time points analysed and abolished the characteristic rhythm in the transcriptional expression of per mRNA. The effect of the per gene silencing in the expression profiles at the transcriptional level of CPR and CYP4EM7 genes showed the loss of rhythmicity and demonstrated the biological clock involvement in the regulation of t heir expression.

GPCR-MAPK signaling pathways underpin fitness trade-offs in whitefly.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Effect of CO binding to P450 BM3 F393 mutants on electron density distribution in the heme cofactor.

Resonance Raman spectroscopy has been performed on a set of cytochrome P450 BM3 heme domains in which mutation of the highly conserved Phe393 induces significant variation in heme iron reduction potential. In previous work [Chen, Z., Ost, T.W.B., and Schelvis, J.P.M. (2004) Biochemistry 43, 1798-1808], a correlation between heme vinyl conformation and the heme iron reduction potential indicated a steric control by the protein over the distribution of electron density in the reduced heme cofactor. The current study aims to monitor changes in electron density on the ferrous heme cofactor following CO binding. In addition, ferric-NO complexes have been studied to investigate potential changes to the proximal Cys400 thiolate. We find that binding of CO to the ferrous heme domains results in a reorientation of the vinyl groups to a largely out-of-plane conformation, the extent of which correlates with the size of the residue at position 393. We conclude that FeII dπ back bonding to the CO ligand largely takes away the need for conjugation of the vinyl groups with the porphyrin ring to accommodate FeII dπ back bonding to the porphyrin ligand. The ferrous-CO and ferric-NO data are consistent with a small decrease in σ-electron donation from the proximal Cys400 thiolate in the F393A mutant and, to a lesser extent, the F393H mutant, potentially due to a small increase in hydrogen bonding to the proximal ligand. Phe393 seems strategically placed to preserve robust σ-electron donation to the heme iron and to fine-tune its electron density by limiting vinyl group rotation.

Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae.

Betanin is a water-soluble red-violet pigment belonging to the betacyanins family. It has become more and more attractive for its natural food colorant properties and health benefits. However, the commercial production of betanin, typically extracted from red beetroot, faces economic and sustainability challenges. Microbial heterologous production therefore offers a promising alternative. Here, we performed combinatorial engineering of plant P450 enzymes and precursor metabolisms to improve the de novo production of betanin in Saccharomyces cerevisiae. Semirational design by computer simulation and molecular docking was used to improve the catalytic activity of CYP76AD. Alanine substitution and site-directed saturation mutants were screened, with a combination mutant showing an approximately 7-fold increase in betanin titer compared to the wild type. Subsequently, betanin production was improved by enhancing the l-tyrosine pathway flux and UDP-glucose supply. Finally, after optimization of the fermentation process, the engineered strain BEW10 produced 134.1 mg/L of betanin from sucrose, achieving the highest reported titer of betanin in a shake flask by microbes. This work shows the P450 enzyme and metabolic engineering strategies for the efficient microbial production of natural complex products.