Cytochrome P450 Genes Research Articles

Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

Phylogenomics of the Ecdysteroid Kinase-like (EcKL) Gene Family in Insects Highlights Roles in Both Steroid Hormone Metabolism and Detoxification.

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid molting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown, and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and we also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. Our evolutionary analyses provide hypotheses of function and a robust framework for future experimental studies of the EcKL gene family. They also open promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied coevolution of butterflies with their host plants.

Hordeum vulgareCYP76M57 catalyzes C2 shortening of tryptophan side chain by C-N bond rearrangement in gramine biosynthesis.

The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cβ. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.

Identification of the chaA and fwA Spore Color Genes of Aspergillus nidulans.

Wild-type Aspergillus nidulans asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the genes of which are dispersed in the genome. The backbone molecule of the pigment is a polyketide synthesized by a polyketide synthase encoded by the wA gene. If wA is not functional, the conidia are white. The polyketide is modified by a laccase encoded by the yA gene and inactivation of yA in an otherwise wild-type background results in yellow spores. Additional spore color mutations have been isolated and mapped to a locus genetically, but the genes that correspond to these loci have not been determined. Spore color markers have been useful historically, and they remain valuable in the molecular genetics era. One can determine if a transforming fragment has been successfully integrated at the wA or yA locus by simply looking at the color of transformant conidia. The genes of the potentially useful color loci chaA (chartreuse conidia) and fwA (fawn conidia) have not been identified previously. We chose a set of candidate genes for each locus by comparing the assembled genome with the genetic map. By systematically deleting these candidate genes, we identified a cytochrome P450 gene (AN10028) corresponding to chaA. Deletions of this gene result in chartreuse conidia and chartreuse mutations can be complemented in trans by a functional copy of this gene. With fwA, we found that the existing fawn mutation, fwA1, is a deletion of 2241 base pairs that inactivates three genes. By deleting each of these genes, we determined that fwA is AN1088, an EthD domain protein. Deletion of AN1088 results in fawn conidia as expected. Neither deletion of chaA nor fwA restricts growth and both should be valuable target loci for transformations. Combinations of deletions have allowed us to investigate the epistasis relationships of wA, yA, chaA and fwA.

Integrated Genome Sequencing and Transcriptome Analysis Identifies Candidate Pathogenicity Genes from Ustilago crameri.

Ustilago crameri is a pathogenic basidiomycete fungus that causes foxtail millet kernel smut (FMKS), a devastating grain disease in most foxtail-millet-growing regions of the world. Here, we report an assembled high-quality genome sequence of U. crameri strain SCZ-6 isolated from the diseased grains of foxtail millet in Changzhi, Shanxi Province, China. The genome size is 19.55 Mb, consisting of 73 contigs (N50 = 840,209 bp) with a G + C content of 54.09%, and encoding 6576 predicted genes and 6486 genes supported by RNA-seq. Evolutionarily, U. crameri lies close to the barley smut U. hordei, and an obvious co-linearity was observed between these two smut fungi. We annotated the genome of U. crameri strain SCZ-6 using databases, identifying 1827 pathogen-host interaction (PHI)-associated genes, 1324 genes encoding fungal virulence factors, 259 CAZy-related genes, 80 genes encoding transporters, and 206 putative cytochrome P450 genes; their expression profiles at different inoculation time points were also detected. Additionally, 70 candidate pathogen effectors were identified according to their expression patterns and predicted functions. In summary, our results provide important insights into the pathogenic mechanisms of the pathogenesis-related genes of U. crameri and a robust foundation for further investigation.

Juvenile hormone III induction reveals key genes in general metabolism, pheromone biosynthesis, and detoxification in Eurasian spruce bark beetle

IntroductionIn recent years, bark beetle Ips typographus, has caused extensive damage to European Norway spruce forests through widespread outbreaks. This pest employs pheromone-assisted aggregation to overcome tree defense, resulting in mass attacks on host spruce. Many morphological and behavioral processes in I. typographus are under the regulation of juvenile hormone III (JH III), including the biosynthesis of aggregation pheromones and associated detoxification monoterpene conjugates.Objectives and MethodsIn this study, we topically applied juvenile hormone III (JH III) and performed metabolomics, transcriptomics, and proteomics in I. typographus both sexes, with focused aims; 1. Highlight the JH III-regulated metabolic processes; 2. Identify pheromone biosynthesis-linked genes; and 3. Investigate JH III’s impact on detoxification conjugates linked to pheromonal components.ResultsNumerous gene families were enriched after JH III treatment, including genes associated with catalytic and oxidoreductase activity, esterases, phosphatases, and membrane transporters. Sex-specific enrichments for reproduction-related and detoxification genes in females and metabolic regulation genes in males were observed. On the protein level were enriched metal ion binding and transferase enzymes in male beetles. After JHIII treatment, mevalonate pathway genes, including terminal isoprenyl diphosphate synthase (IPDS), were exclusively 35- folds upregulated in males, providing evidence of de novo biosynthesis of pheromone components 2-methyl-3-buten-2-ol and ipsdienol. In addition, cytochrome P450 genes likely involved in the biosynthesis of cis/trans-verbenol, detoxification, and formation of ipsdienol, were 3-fold upregulated in the male gut. The increase in gene expression correlated with the heightened production of the respective metabolites. Detoxification conjugates, verbenyl oleate in the beetle fat body and verbenyl diglycosides in the gut, were induced by JHIII application, which confirms the hormone regulation of their formation. The JH III induction also increased the gene contigs esterase and glycosyl hydrolase up to proteins from male gut tissue. The esterase was proposed to release pheromone cis-verbenol in adult males by breaking down verbenyl oleate. The correlating analyses confirmed a reduction in the abundance of verbenyl oleate in the induced male beetle.ConclusionThe data provide evidence of JH III’s regulatory role in the expression of genes and enzymes related to fundamental beetle metabolism, pheromone biosynthesis, and detoxification in Ips typographus.

Genomic basis of seed colour in quinoa inferred from variant patterns using extreme gradient boosting.

Quinoa is an agriculturally important crop species originally domesticated in the Andes of central South America. One of its most important phenotypic traits is seed colour. Seed colour variation is determined by contrasting abundance of betalains, a class of strong antioxidant and free radicals scavenging colour pigments only found in plants of the order Caryophyllales. However, the genetic basis for these pigments in seeds remains to be identified. Here we demonstrate the application of machine learning (extreme gradient boosting) to identify genetic variants predictive of seed colour. We show that extreme gradient boosting outperforms the classical genome-wide association approach. We provide re-sequencing and phenotypic data for 156 South American quinoa accessions and identify candidate genes potentially controlling betalain content in quinoa seeds. Genes identified include novel cytochrome P450 genes and known members of the betalain synthesis pathway, as well as genes annotated as being involved in seed development. Our work showcases the power of modern machine learning methods to extract biologically meaningful information from large sequencing data sets.

Cytochrome P450 genes expression in human prostate cancer

CYP-dependent metabolites play a critical role in regulating the cell cycle, as well as the proliferative, invasive, and migratory activity of cancer cells. We conducted a study to analyze the relative gene expression of various CYPs (CYP7B1, CYP27A1, CYP39A1, CYP51, CYP1B1, CYP3A5, CYP4F8, CYP5A1, CYP4F2, CYP2J2, CYP2E1, CYP2R1, CYP27B1, CYP24A1) in 41 pairs of prostate samples (tumor and conventional normal tissues) using qPCR. Our analysis determined significant individual variability in the expression levels of all studied CYPs, both in the tumor and in conventionally normal groups. However, when we performed a paired test between the tumor and normal groups, we found no significant difference in the expression of the studied genes. We did observe a tendency to increase the level of CYP1B1 expression in the tumor group. We also did not find any significant difference between the levels of the studied CYPs in the tumor and conventional normal groups at different stages of prostate cancer and pathomorphological indicators. Correlation analysis revealed the presence of a positive relationship between the expressions of some cholesterol-metabolizing CYP genes, as well as between genes responsible for vitamin D biosynthesis and cholesterol biosynthesis. We observed significant correlative relationships between the expression of CYPs and some prostate cancer-related genes (CDH2, MMP9, SCHLAP1, GCR, CYP17A1, ACTA2, CXCL14, FAP, CCL17, MSMB, IRF1, VDR). Therefore, the expression of CYPs is not directly associated with prostate cancer but is largely determined by genetic, epigenetic factors, as well as endogenous substrates and xenobiotics. The significant correlative relationship between CYPs and genes associated with cancer may indicate common regulatory pathways that may have a synergistic effect on the tumor, ensuring the survival of cancer cells.

Signatures of Co-evolution and Co-regulation in the CYP3A and CYP4F Genes in Humans.

Cytochromes P450 (CYP450) are hemoproteins generally involved in the detoxification of the body of xenobiotic molecules. They participate in the metabolism of many drugs and genetic polymorphisms in humans have been found to impact drug responses and metabolic functions. In this study, we investigate the genetic diversity of CYP450 genes. We found that two clusters, CYP3A and CYP4F, are notably differentiated across human populations with evidence for selective pressures acting on both clusters: we found signals of recent positive selection in CYP3A and CYP4F genes and signals of balancing selection in CYP4F genes. Furthermore, an extensive amount of unusual linkage disequilibrium is detected in this latter cluster, indicating co-evolution signatures among CYP4F genes. Several of the selective signals uncovered co-localize with expression quantitative trait loci (eQTL), which could suggest epistasis acting on co-regulation in these gene families. In particular, we detected a potential co-regulation event between CYP3A5 and CYP3A43, a gene whose function remains poorly characterized. We further identified a causal relationship between CYP3A5 expression and reticulocyte count through Mendelian randomization analyses, potentially involving a regulatory region displaying a selective signal specific to African populations. Our findings linking natural selection and gene expression in CYP3A and CYP4F subfamilies are of importance in understanding population differences in metabolism of nutrients and drugs.

Placental cytochrome P450 methylomes in infants exposed to prenatal opioids: exploring the effects of neonatal opioid withdrawal syndrome on health horizons.

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights. Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications. Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers. Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances. Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment.

Association of hard fescue (Festuca brevipila) stress tolerances with genome mapped markers

AbstractHard fescue is an allo‐hexaploid, caespitose, cool‐season grass that is part of the fine‐leaved ovina complex of the Lolium–Festuca clade in Poaceae. It is predominantly used as a low‐maintenance turfgrass and soil stabilization grass in situations with reduced inputs of nitrogen and water. While often recommended for use under tree canopies, prolonged foliar shade can thin turf stands and reduce their functionality; furthermore, gray snow mold caused by Typhula incarnata can further affect hard fescue turfgrass functionality. In this study, we gathered a diverse population of hard fescue collections and varieties, which fit into four genetic structures. Because no genomic reference sequences exist for this species, a contig‐level pseudohaploid genomic reference was assembled from a plant of the hard fescue variety Beacon. With 98% benchmarking universal single‐copy orthologs (BUSCO) coverage from 241 contigs, this reference assembly was used to conduct genome‐wide association analysis. Thirty‐eight single‐nucleotide polymorphism (SNP) markers were associated with foliar shade response phenotypes, including SNPs near molybdate transporter and cytochrome P450 genes. Three SNP markers were associated with gray snow mold responses, with one located near a Protein Kinase G11 gene and corresponding to a 20% improvement in gray snow mold resistance.

Analysis of Genes Associated with Feeding Preference and Detoxification in Various Developmental Stages of Aglais urticae.

Herbivorous insects and host plants have developed a close and complex relationship over a long period of co-evolution. Some plants provide nutrients for insects, but plants' secondary metabolites also influence their growth and development. Urtica cannabina roots and leaves are poisonous, yet Aglais urticae larvae feed on them, so we aimed to clarify the mechanism enabling this interaction. At present, studies on the detoxification mechanism of the A. urticae are rare. In our study, first, we used the A. urticae larval odor selection behavior bioassay and choice feeding preference assay to analyze the feeding preferences of A. urticae on its host plant, U. cannabina. Next, we used transcriptome sequencing to obtain the unigenes annotated and classified by various databases, such as KEGG and GO. In this study, we found that U. cannabina could attract A. urticae larvae to feed via scent, and the feeding preference assay confirmed that larvae preferred U. cannabina leaves over three other plants: Cirsium japonicum, Cannabis sativa, and Arctium lappa. The activity of detoxifying enzymes GST and CarE changed in larvae that had consumed U. cannabina. Furthermore, through transcriptomic sequencing analysis, 77,624 unigenes were assembled from raw reads. The numbers of differentially expressed genes were calculated using pairwise comparisons of all life stages; the expression of detoxification enzyme genes was substantially higher in larvae than in the pupal and adult stages. Finally, we identified and summarized 34 genes associated with detoxification enzymes, such as UDP-glucose 4-epimerase gene, 5 Glutathione S-transferase genes, 4 Carboxylesterase genes, 4 Cytochrome P450 genes, 10 ATP-binding cassette genes, 4 Superoxide dismutase, and Peroxidase. Moreover, we identified 28 genes associated with the development of A. urticae. The qRT-PCR results were nearly consistent with the transcriptomic data, showing an increased expression level of four genes in larvae. Taken together, this study examines the correlation between A. urticae and host plants U. cannabina, uncovering a pronounced preference for A. urticae larvae toward host plants. Consistent with RNA-seq, we investigated the mechanism of A. urticae's interaction with host plants and identified detoxification-related genes. The present study provides theoretical support for studying insect adaptation mechanisms and biological control.

Polymorphism of xenobiotic biotransformation enzyme genes and their role in early pregnancy loss

Considering the progressive decrease in the number of women of fertile age, as well as children and adolescents, the primary medical and social task at the present stage is to preserve the reproductive health and life of women, preserving every desired pregnancy. The concept of demographic policy of the Russian Federation for the period until 2025 defines the global national goals, i.e. to increase the total fertility rate by 1.5 times, to reduce maternal and infant mortality by at least 2 times, and to improve the reproductive health of women, children and adolescents. Today every fifth desirable pregnancy ends in early loss, and the rate of miscarriage does not tend to decrease and, in fact, increases every year. Carriage of susceptibility or candidate genes can alter the course of biochemical processes in women and cause early pregnancy losses. According to the literature, the cytochrome P-450 gene (CYP1A1, CYP1B1), xenobiotic detoxification genes (GSTT1, GSTM1, GSTP1) and many others are recognized as candidate genes (genetic markers) associated with reproductive losses. Adverse pregnancy outcome is usually multifactorial; in turn, the combination of polymorphic variants of different candidate genes can increase the risk of early losses. A detailed study of the role of candidate genes with clarity and confirmation of the interest of candidate genes in the development of pregnancy loss is undeniable. In this regard, the interest of many researchers in studying the polymorphism of genes of the xenobiotic biotransformation enzyme system as one of the causal factors of pregnancy failure is understandable. The study of this group of genes is of particular relevance in regions with developed industry and high anthropogenic load. The article is devoted to the analysis of the existing data of foreign and domestic literature sources concerning the relationship between polymorphism of xenobiotic biotransformation system genes and reproductive losses.

The telomere-to-telomere (T2T) genome of Peucedanum praeruptorum Dunn provides insights into the genome evolution and coumarin biosynthesis.

Traditional Chinese medicine has used Peucedanum praeruptorum Dunn (Apiaceae) for a long time. Various coumarins, including the significant constituents praeruptorin (A-E), are the active constituents in the dried roots of P. praeruptorum. Previous transcriptomic and metabolomic studies have attempted to elucidate the distribution and biosynthetic network of these medicinal-valuable compounds. However, the lack of a high-quality reference genome impedes an in-depth understanding of genetic traits and thus the development of better breeding strategies. A telomere-to-telomere (T2T) genome was assembled for P. praeruptorum by combining PacBio HiFi, ONT ultra-long, and Hi-C data. The final genome assembly was approximately 1.798 Gb, assigned to 11 chromosomes with genome completeness >98%. Comparative genomic analysis suggested that P. praeruptorum experienced 2 whole-genome duplication events. By the transcriptomic and metabolomic analysis of the coumarin metabolic pathway, we presented coumarins' spatial and temporal distribution and the expression patterns of critical genes for its biosynthesis. Notably, the COSY and cytochrome P450 genes showed tandem duplications on several chromosomes, which may be responsible for the high accumulation of coumarins. A T2T genome for P. praeruptorum was obtained, providing molecular insights into the chromosomal distribution of the coumarin biosynthetic genes. This high-quality genome is an essential resource for designing engineering strategies for improving the production of these valuable compounds.

Pigmentation of soybean seed coats via a mutation that abolishes production of multiple-phased siRNAs of chalcone synthase genes.

Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.

Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Gemcitabine and synthesized silver nanoparticles impact on chemically induced hepatocellular carcinoma in male rats.

Objective: Gemcitabine (GEM) is a deoxycytidine analog chemotherapeutic drug widely used to treat many cancers. Silver nanoparticles (AgNPs) are important nanomaterials used to treat many diseases. Using gamma radiation in nanoparticle preparation is a new eco-friendly method. This study aims to evaluate the efficiency of co-treating gemcitabine and silver nanoparticles in treating hepatocellular carcinoma. Method: The AgNPs were characterized using UV-visible spectroscopy, XRD, TEM, and EDX. The MTT cytotoxicity in vitro assay of gemcitabine, doxorubicin, and cyclophosphamide was assessed against Wi38 normal fibroblast and HepG2 HCC cell lines. After HCC development, rats received (10µg/g b.wt.) of AgNPs three times a week for 4weeks and/or GEM (5mg/kg b.wt.) twice weekly for 4weeks. Liver function enzymes were investigated. Cytochrome P450 and miR-21 genes were studied. Apoptosis was determined by using flow cytometry, and apoptotic modifications in signaling pathways were evaluated via Bcl-2, Bax, Caspase-9, and SMAD-4. Results: The co-treatment of GEM and AgNPs increased apoptosis by upregulating Bax and caspase 9 while diminishing Bcl2 and SMAD4. It also improved cytochrome P450 m-RNA relative expression. The results also proved the cooperation between GEM and AgNPs in deactivating miR21. The impact of AgNPs as an adjuvant treatment with GEM was recognized. Conclusions: The study showed that co-treating AgNPs and GEM can improve the efficiency of GEM alone in treating HCC. This is achieved by enhancing intrinsic and extrinsic apoptotic pathways while diminishing some drawbacks of using GEM alone.

The role of polymorphic cytochrome P450 gene (CYP2B6) in B-chronic lymphocytic leukemia (B-CLL) incidence and outcome among Egyptian patients.

Cytochromes P450 (CYPs) play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75% of the total metabolism of commercially available drugs, including chemotherapeutics. The gene expression and enzyme activity of CYPs are variable between individuals, which subsequently leads to different patterns of susceptibility to carcinogenesis by genotoxic xenobiotics, as well as differences in the efficacy and toxicity of clinically used drugs. This research aimed to examine the presence of the CYP2B6*9 polymorphism and its possible association with the incidence of B-CLL in Egyptian patients, as well as the clinical outcome after receiving cyclophosphamide chemotherapy. DNA was isolated from whole blood samples of 100 de novo B-CLL cases and also from 100 sex- and age-matched healthy individuals. The presence of the CYP2B6*9 (G516T) polymorphism was examined by PCR-based allele specific amplification (ASA). Patients were further indicated for receiving chemotherapy, and then they were followed up. The CYP2B6*9 variant indicated a statistically significant higher risk of B-CLL under different genetic models, comprising allelic (T-allele vs. G-allele, OR = 4.8, p < 0.001) and dominant (GT + TT vs. GG, OR = 5.4, p < 0.001) models. Following cyclophosphamide chemotherapy, we found that the patients with variant genotypes (GT + TT) were less likely to achieve remission compared to those with the wild-type genotype (GG), with a response percentage of (37.5% vs. 83%, respectively). In conclusion, our findings showed that the CYP2B6*9 (G516T) polymorphism is associated with B-CLL susceptibility among Egyptian patients. This variant greatly affected the clinical outcome and can serve as a good therapeutic marker in predicting response to cyclophosphamide treatment.

Genome wide and comprehensive analysis of the cytochrome P450 (CYPs) gene family in Pyrus bretschneideri: Expression patterns during Sporidiobolus pararoseus Y16 enhanced with ascorbic acid (VC) treatment

Cytochrome P450s (CYPs) constitute the largest group of enzymes in plants and are involved in a variety of processes related to growth and protection. However, the CYP gene superfamily in pear (Pyrus bretschneideri) and their characteristics is unclear. Through a comprehensive genome-wide analysis, this article identified a total of 74 CYP genes in the P. bretschneideri genome, which were categorized into fourteen families. Motif analysis reveals that most of the ten motifs predicted were with the p450 conserved domain. The majority of the CYP genes have exon arrangements. Furthermore, promoter analysis unveiled a multitude of cis-acting elements associated with diverse responsiveness including hormones, light responsive, anoxic specific inducibility and anaerobic induction. Analysis of the transcriptome data reveal that about 80% of the pear CYPs genes were upregulated and they were positively correlated with the antioxidant's parameters such as total flavonoids and total phenol content as well as ABTS and DPPH radicals. RT-qPCR analysis confirmed that the CYP genes could be regulated in pear. Collectively, our results reveal comprehensive insights into the CYP superfamily in pear and make a valuable contribution to the ongoing process of functional validation.