Target Site Research Articles

Application of CRISPR/Cas9 gene editing technology in edible fungi: a review

The CRISPR/Cas9 gene editing system is a versatile technology for modifying gene, playing a crucial role in the study of functional genes and genetic breeding of plants, animals, fungi, and microorganisms. This review provides a comprehensive analysis of the application of this technology in gene research and genetic breeding of edible fungi. The review covers various aspects, including the delivery and expression strategies of Cas9 and sgRNA, genetic transformation methods, mutant screening, and repair strategies for target sites following DNA double-strand breaks. Additionally, the review summarizes the main challenges and optimization strategies associated with the application of this technology in edible fungi. Lastly, the future application potential of this technology in edible fungi research is discussed, drawing from the authors' personal research background.

Survey of target site mutations linked with insecticide resistance in Italian populations of Aphis gossypii.

Aphis gossypii is a worldwide agricultural pest that causes high levels of economic losses by feeding and transmitting virus diseases. It is usually controlled by chemical insecticides, but this could lead to the selection of resistant populations. Several single nucleotide polymorphisms (SNPs) have been identified associated with insecticide resistance. Monitoring activities to detect the presence of such mutations in field populations can have an important role in insect pest management but, currently, no information on Italian strains is available. The presence of target site mutations conferring resistance to different insecticides was analysed in Italian field collected populations of A. gossypii with an allele specific approach (QSGG, Qualitative Sybr-Green Genotyping). Primers were designed to detect mutations in genes coding acetylcholinesterase (S431F), nicotinic acetylcholine receptor (R81T) and voltage-gated sodium channel (M918L and L1014F). S431F was widespread but with high variability across populations. R81T was detected for the first time in Italy but only in two populations. The L1014F mutation (kdr) was not found, while in the samples showing the M918L two different nucleotidic substitutions were detected. Mutant allele frequencies were, respectively, 0.70 (S431), 0.31 (M918) and 0.02 (R81). Further analysis on the voltage-gated sodium channel gene showed the presence of eight haplotypes and one non-synonymous mutation in the gene coding region. Multiple target-site mutations were detected within Italian populations. The combinations of genotypes observed in certain locations could affect negatively the control of this pest. Preliminary insights on the genetic structure in the Italian populations of A. gossypii were acquired. © 2024 Society of Chemical Industry.

Fast Track Adaptation of Oncolytic Coxsackie B3 Virus to Resistant Colorectal Cancer Cells - a Method to Personalize Virotherapy

BackgroundThe efficacy of oncolytic viruses (OV) in cancer treatment depends on their ability to successfully infect and destroy tumor cells. However, patients’ tumors vary, and in the case of individual insensitivity to an OV, therapeutic efficacy is limited. Here, we present a protocol for rapid generation of tumor cell-specific adapted oncolytic coxsackievirus B3 (CVB3) with enhanced oncolytic potential and a satisfactory safety profile. This is achieved by combining directed viral evolution (DVE) with genetic modification of the viral genome and the use of a microRNA-dependent regulatory tool.MethodsThe oncolytic CVB3 variant PD-H was adapted to the refractory colorectal carcinoma cell line Colo320 through serial passaging. XTT assays and virus plaque assays were used to determine virus cytotoxicity and virus replication in vitro. Recombinant PD-H variants were generated through virus mutagenesis. Apoptosis was detected by Western blots, Caspase 3/7 assays, and DAPI staining. The therapeutic efficacy and safety of the adapted recombinant OV PD-SK-375TS were assessed in vivo using a subcutaneous Colo320 xenograft mouse model.ResultsPD-H was adapted to the colorectal cancer cell line Colo320 within 10 passages. Sequencing of passage 10 virus P-10 revealed a heterogenous virus population with five nucleotide mutations resulting in amino acid substitutions. The genotypically homogeneous OV PD-SK was generated by inserting the five detected mutations of P-10 into the genome of PD-H. PD-SK showed significantly stronger replication and cytotoxicity than PD-H in Colo320 cells, but not in other colorectal carcinoma cell lines. Increase of apoptosis induction was detected as key mechanisms of Colo320 cell-specific adaptation of PD-SK. For in vivo safety PD-SK was engineered with target sites of the miR-375 (miR-375TS) to exclude virus replication in normal tissues. PD-SK-375TS, unlike the PD-H-375TS not adapted homolog suppressed the growth of subcutaneous Colo320 tumors in nude mice without causing any side effects.ConclusionTaken together, here we present an optimized protocol for the rapid generation of tumor cell-specific adapted oncolytic CVB3 based on the oncolytic CVB3 strain PD-H. The protocol is promising for the generation of personalized OV for tumor therapy and has the potential to be applied to other OV.

Appraisal of the Neuroprotective Potentials of Isoeugenol Using In-vitro, In-vivo and In-silico Approaches

Appraisal of the Neuroprotective Potentials of Isoeugenol Using In-vitro, In-vivo and In-silico Approaches

Structural Basis for the Interaction of Redβ Single-Strand Annealing Protein with Escherichia coli Single-Stranded DNA-Binding Protein

Redβ is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redβ is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redβ can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redβ with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redβ and SSB involves the C-terminal domain (CTD) of Redβ and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redβ CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redβ and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redβ-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.

Investigating the interplay of genetic variations, MCP-1 polymorphism, and docking with phytochemical inhibitors for combatting dengue virus pathogenicity through in silico analysis

Abstract Understanding ten significant of dengue virus a paramount due to its persistent threat across the globe causing numerous epidemic and millions of deaths every year. Despite advancement in healthcare, emerging outbreaks continue to claim millions of lives annually. The virus with its various serotype possesses a significant challenge to public health worldwide, its transmission through the arthropods which feed on the blood of higher animals further exacerbates its impact. This elucidating the mechanism and factor contributing to dengue virus pathogenicity is essential for developing strategies to combat its spreading. A systemic review is done while studying about Dengue virus serotypes to evaluate the relationship of dengue with other viral load and to develop an inhibitor in viral protein by using different bioinformatics tools in silico molecular docking. Phytochemicals were chosen to hit the target site of protein for inhibiting its active site in pathogenesis of Dengue virus. A phylogenetic lineage was observed of virus with other members of its family. Dengue virus has same similarities in some part of genomic structure with other viruses. Data bases were used to trace the genome of the virus. Different components of virus were analyzed and link was developed among those components within virus and other family members. Protein docking was performed by using the bioinformatics software auto docking. A pharmaceutical drug designing was developed to create inhibitor of protein structure in Dengue virus serotype-2. A comprehensive review of the methodology employed in our study including the experimental and computational techniques were utilized. Further we presented the results by analysis of genetic variation MCP-1 polymorphism and docking phytochemical inhibitors.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization.

YY1 binding is a gene-intrinsic barrier to Xist-mediated gene silencing

X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. The chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as a barrier to Xist-mediated silencing until the late stages of the XCI process.

Reduced Prevalence of Parkinson's Disease in Patients Prescribed Calcineurin Inhibitors.

Preclinical evidence suggests calcineurin inhibitors (CNIs) combat α-synuclein-induced neuronal dysfunction and motor impairments. However, whether CNIs prevent or treat Parkinson's disease (PD) in humans has never been investigated. We seek to ascertain if prescription of CNIs is linked to a decreased prevalence of PD in a varied patient population and to glimpse into the mechanism(s) and target site through which CNIs might decrease PD prevalence. We analyzed electronic health records (EHRs) from patients prescribed the brain penetrant CNI tacrolimus (TAC), the peripherally restricted CNI cyclosporine (CySp), or the non-CNI sirolimus (SIR). For comparison, EHRs from a diverse population from the same network served as a general population-like control. After propensity-score matching, prevalence, odds, and hazards of PD diagnoses among these cohorts were compared. Patients prescribed CNIs have decreased odds of PD diagnosis compared to the general population-like control, while patients prescribed SIR do not. Notably, patients prescribed TAC have a decreased prevalence of PD compared to patients prescribed SIR or CySp. Our results suggest CNIs, especially those acting within the brain, may prevent PD. The reduced prevalence of PD in patients prescribed TAC, compared to patients prescribed SIR, suggests that mechanisms of calcineurin inhibition- other than immunosuppression, which is common to both drugs- are driving the reduction. Therefore, CNIs may provide a promising therapeutic approach for PD.

Cinnamaldehyde: An effective component of Cinnamomum cassia inhibiting Helicobacter pylori

Ethnopharmacological relevanceCinnamomum cassia Presl (Cinnamomum cassia) is a common traditional Chinese medicine, which can promote the secretion and digestion of gastric juice, improve the function of gastrointestinal tract. Cinnamaldehyde (CA) is a synthetic food flavoring in the Chinese Pharmacopoeia. Aim of the studyThis study aimed to search for the active ingredient (CA) of inhibiting H. pylori from Cinnamomum cassia, and elucidate mechanism of action, so as to provide the experimental basis for the treatment of H. pylori infection with Cinnamomum cassia. Materials and methodsIt's in vitro and in vivo pharmacological properties were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and an acute gastric inflammation model in mice infected with H. pylori. Drug safety was evaluated using the CCK8 method and high-dose administration in mice. The advantageous characteristics of CA in inhibiting H. pylori were confirmed using acidic conditions and in combination with the antibiotics. The mechanism underlying the action of CA on H. pylori was explored using scanning electron microscopy (SEM), adhesion experiments, biofilm inhibition tests, ATP and ROS release experiments, and drug affinity responsive target stability (DARTS) screening of target proteins. The protein function and target genes were verified by molecular docking and Real-Time quantitative reverse transcription PCR (qRT-PCR). ResultsThe results demonstrated that CA was found to be the main active ingredient against H. pylori in Cinnamomum cassia in-vitro tests, with a MIC of 8–16 μg/mL. Moreover, CA effectively inhibited both sensitive and resistant H. pylori strains. The dual therapy of PPI + CA exhibited remarkable in vivo efficacy in the acute gastritis mouse model, superior to the standard triple therapy. DARTS, molecular docking, and qRT-PCR results suggested that the target sites of action were closely associated with GyrA, GyrB, AtpA, and TopA, which made DNA replication and transcription impossible, then leading to inhibition of bacterial adhesion and colonization, suppression of biofilm formation, and inhibition ATP and enhancing ROS. ConclusionsThis study demonstrated the suitability of CA as a promising lead drug against H. pylori, The main mechanisms can target GyrA ect, leading to reduce ATP and produce ROS, which induces the apoptosis of bacterial.

Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, β-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

Transduction and Genome Editing of the Heart with Adeno-Associated Viral Vectors Loaded onto Electrospun Polydioxanone Nonwoven Fabrics.

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.

Ash seedlings in a reciprocal transplant experiment—the extent of damage of mature forest stands affects ash offspring performance

In past decades, ash dieback has caused a rapid decline of European ash (Fraxinus excelsior) in temperate forests of Europe. Numerous studies focus on mitigating the negative impacts of ash dieback to forest ecosystems or identifying resistant genotypes. The role of natural selection toward genotypes withstanding ash dieback for ash regeneration has been less frequently studied with experimental means to date. This is, however, necessary in times of global change, because the preservation of ash in Europe’s forests will depend, above all, on the adaptability of the future generations of ash trees. To quantify the extent and effects of ash dieback severity for ash regeneration we selected five forest stands moderately damaged and five forest stands highly damaged by ash dieback, in Schleswig-Holstein, Germany. We reciprocally transplanted naturally regenerated ash seedlings sampled in the field between these 10 sites. A shading treatment added to each half of the plots per site was meant to test for effects of altered light conditions in the herb layer due to canopy opening caused by ash dieback. With this approach, we tested seedling survival, performance and fungal infection for an interacting effect of origin and target site in regard to ash dieback severity and environmental factors over 2 years and recorded leaf traits (specific leaf area, leaf dry matter content) in the second year. Reduced light conditions under the shading nets had strong effects, influencing first year performance and infection probability as well as second year survival, growth and leaf trait characteristics. Soil conditions had only a marginal influence on transplanted seedlings. Transplantation direction between moderately and highly damaged sites affected infection marginally during the first year and survival as well as leaf traits significantly during the second year. Most notably, seedlings transplanted from moderately damaged to severely damaged sites exhibited the highest infection probability and lowest SLA, while seedlings transplanted vice versa were least likely to be infected and exhibited the highest SLA. Results hint at a first filtering effect by the ash dieback history of a forest stand and might indicate a transition from ecologically to evolutionary driven differentiation of ash seedling responses.

Tracing toxic path of antimony: From bioaccumulation to DNA hypomethylation in zebrafish (Danio rerio)

The increasing concentration of Antimony (Sb) in ecological environments has raised serious concerns about its potential biotoxicological impact. This study investigated the toxicokinetics, Global DNA Methylation (GDM), biomarker expression, and Integrated Biological Response (IBR) of Sb at different concentrations in zebrafish. The toxic mechanism of Sb exposure was simulated using molecular dynamics (MD). The results showed that significant differences effect existed (BCFk: liver > ovary > gut > brain) and uptake saturation phenomenon of Sb among zebrafish tissues. Over a 54-day exposure period, the liver emerged as the main target site for Sb-induced GDM, and the restoration was slower than in other tissues during the 54-day recovery period. Moreover, the concentration of Sb had a significant impact on the normally expression of biomarkers, with GSTM1 inhibited and MTF2, MT1, TET3, and p53 showing varying degrees of activation at different Sb concentrations. This could be attributed to Sb3+ potentially occupying the active site or tightly binding to the deep cavity of these genes. The IBR and MD results highlighted DNMT1 as the most sensitive biomarker among those assessed. This heightened sensitivity can be attributed to the stable binding of Sb3+ to DNMT1, resulting in alterations in the conformation of DNMT1's catalytic domain and inhibition of its activity. Consequently, this disruption leads to damage to the integrity of GDM. The study suggests that DNA methylation could serve as a valuable biomarker for assessing the ecotoxicological impact of Sb exposure. It contributes to a better understanding of the toxicity mechanisms in aquatic environments caused potential pollutants.

How to use CRISPR/Cas9 in plants - from target site selection to DNA repair.

A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Inhibiting the cGAS-STING Pathway in Ulcerative Colitis with Programmable Micelles.

Ulcerative colitis is a chronic condition in which a dysregulated immune response contributes to the acute intestinal inflammation of the colon. Current clinical therapies often exhibit limited efficacy and undesirable side effects. Here, programmable nanomicelles were designed for colitis treatment and loaded with RU.521, an inhibitor of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. STING-inhibiting micelles (SIMs) comprise hyaluronic acid-stearic acid conjugates and include a reactive oxygen species (ROS)-responsive thioketal linker. SIMs were designed to selectively accumulate at the site of inflammation and trigger drug release in the presence of ROS. Our in vitro studies in macrophages and in vivo studies in a murine model of colitis demonstrated that SIMs leverage HA-CD44 binding to target sites of inflammation. Oral delivery of SIMs to mice in both preventive and delayed therapeutic models ameliorated colitis's severity by reducing STING expression, suppressing the secretion of proinflammatory cytokines, enabling bodyweight recovery, protecting mice from colon shortening, and restoring colonic epithelium. In vivo end points combined with metabolomics identified key metabolites with a therapeutic role in reducing intestinal and mucosal inflammation. Our findings highlight the significance of programmable delivery platforms that downregulate inflammatory pathways at the intestinal mucosa for managing inflammatory bowel diseases.

TEC-miTarget: enhancing microRNA target prediction based on deep learning of ribonucleic acid sequences

BackgroundMicroRNAs play a critical role in regulating gene expression by binding to specific target sites within gene transcripts, making the identification of microRNA targets a prominent focus of research. Conventional experimental methods for identifying microRNA targets are both time-consuming and expensive, prompting the development of computational tools for target prediction. However, the existing computational tools exhibit limited performance in meeting the demands of practical applications, highlighting the need to improve the performance of microRNA target prediction models.ResultsIn this paper, we utilize the most popular natural language processing and computer vision technologies to propose a novel approach, called TEC-miTarget, for microRNA target prediction based on transformer encoder and convolutional neural networks. TEC-miTarget treats RNA sequences as a natural language and encodes them using a transformer encoder, a widely used encoder in natural language processing. It then combines the representations of a pair of microRNA and its candidate target site sequences into a contact map, which is a three-dimensional array similar to a multi-channel image. Therefore, the contact map's features are extracted using a four-layer convolutional neural network, enabling the prediction of interactions between microRNA and its candidate target sites. We applied a series of comparative experiments to demonstrate that TEC-miTarget significantly improves microRNA target prediction, compared with existing state-of-the-art models. Our approach is the first approach to perform comparisons with other approaches at both sequence and transcript levels. Furthermore, it is the first approach compared with both deep learning-based and seed-match-based methods. We first compared TEC-miTarget’s performance with approaches at the sequence level, and our approach delivers substantial improvements in performance using the same datasets and evaluation metrics. Moreover, we utilized TEC-miTarget to predict microRNA targets in long mRNA sequences, which involves two steps: selecting candidate target site sequences and applying sequence-level predictions. We finally showed that TEC-miTarget outperforms other approaches at the transcript level, including the popular seed match methods widely used in previous years.ConclusionsWe propose a novel approach for predicting microRNA targets at both sequence and transcript levels, and demonstrate that our approach outperforms other methods based on deep learning or seed match. We also provide our approach as an easy-to-use software, TEC-miTarget, at https://github.com/tingpeng17/TEC-miTarget. Our results provide new perspectives for microRNA target prediction.

Genome-wide identification of G-protein coupled receptors (GPCRs) and their expression profile in response to β-cypermethrin stress in Zeugodacus cucurbitae

G-protein coupled receptors (GPCRs) are the largest and most diverse transmembrane receptor family in the cell. They are involved in regulating a wide range of biological processes, including behavior, reproduction, and development. However, GPCRs have not yet been identified in Zeugodacus cucurbitae. The current study focuses on the GPCRs identification, classification, distribution, and their expression analysis under β-cypermethrin stress to uncover novel targets for pest management and assist in the development of effective strategies for controlling the melon fly population. We identified 80 GPCRs genes including 50 GPCRs identified in family A, 17 GPCRs identified in family B, 8 identified in family C, and 5 identified in family F. Z. cucurbitae GPCRs showed significant differences in both the number of genes in families or subfamilies, as well as the sequencing of the genes. Interestingly, newly identified GPCRs genes are expressed differently at various developmental stages of Z. cucurbitae. Further, we evaluated these 80 GPCRs using Realtime quantitative PCR to confirm their expression between β-cypermethrin-resistant (RS) strain and susceptible strain (SS) of Z. cucurbitae. We identified 50 GPCR genes were highly overexpressed in a RS. Among these genes, eight genes were strongly induced by the 30% lethal concentration (LC) while two genes were significantly increased by the 50% LC of β-cypermethrin. This first genome-wide profiling and characterization of GPCRs could lay foundation for unraveling detoxification mechanism and target site modifications which may improve the insect resistance and could be effective insecticide targets for Z. cucurbitae management.

Bulk segregant mapping and transcriptome analyses reveal the molecular mechanisms of spinetoram resistance in Spodoptera frugiperda

The evolution of resistance to insecticides poses a significant threat to pest management programs. Understanding the molecular mechanisms underlying insecticide resistance is essential to design sustainable pest control and resistance management programs. The fall armyworm, Spodoptera frugiperda, is an important insect pest of many crops and has a remarkable ability to evolve resistance to insecticides. In this study, we employed bulk segregant analysis (BSA) combined with DNA and RNA sequencing to characterize the molecular basis of spinetoram resistance in S. frugiperda. Analysis of genomic data derived from spinetoram selected and unselected bulks and the spinetoram-resistant and susceptible parental strains led to the identification of a three-nucleotide deletion in the gene encoding the nicotinic acetylcholine receptor α6 subunit (nAChR α6). Transcriptome profiling identified the upregulation of few genes encoding detoxification enzymes associated with spinetoram resistance. Thus, spinetoram resistance in S. frugiperda appears to be mediated mainly by target site insensitivity with a minor role of detoxification enzymes. Our findings provide insight into the mechanisms underpinning resistance to spinetoram in S. frugiperda and will inform the development of strategies to control this highly damaging, globally distributed crop pest.