Selected Target Genes Research Articles

Betaine enhances SCAPs chondrogenic differentiation and promotes cartilage repair in TMJOA through WDR81

BackgroundThe cartilage tissue regeneration mediated with mesenchymal stem cells (MSCs) is considered as a viable strategy for temporomandibular joint osteoarthritis (TMJOA). Betaine has been confirmed to modulate the multidirectional differentiation of MSCs, while its effect on chondrogenic differentiation of Stem Cells from the Apical Papilla (SCAPs) is unknown. Here, we explored the effects and underlying mechanisms of betaine on chondrogenic differentiation of SCAPs.MethodsBetaine was added for SCAPs chondrogenic induction. The chondrogenic differentiation potential was assessed using Alcian Blue staining, Sirius Red staining and the main chondrogenic markers. In vivo cartilage regeneration effects were evaluated by the rat TMJOA model. RNA-sequencing and biological analyses were performed to select target genes and biological processes involved. The mechanism betaine acts on chondrogenic differentiation of SCAPs was further explored.ResultsBetain-treated SCAPs demonstrated stronger cartilage regeneration in vitro and promoted cartilage repair of TMJOA in vivo. Betaine enhanced the expression of WDR81 in SCAPs during chondrogenesis. WDR81 overexpression promoted chondrogenic differentiation of SCAPs, while WDR81 depletion inhibited chondrogenic differentiation. In addition, both betaine treatment and WDR81 overexpression reduced intracellular reactive oxygen species levels and increased mitochondrial membrane potential in SCAPs.ConclusionBetaine promotes SCAPs chondrogenic differentiation and provided an effective candidate for TMJOA treatment. WDR81 may serve as the potential drug target through mitophagy.

Impact of UV-Irradiated Mesoporous Titania Nanoparticles (mTiNPs) on Key Onco- and Tumor Suppressor microRNAs of PC3 Prostate Cancer Cells

Background: Mesoporous titanium dioxide nanoparticles (mTiNPs) are known for their chemical stability, non-toxicity, antimicrobial and anticancer effects, as well as for their photocatalytic properties. When this material is subjected to UV radiation, its electronic structure shifts, and during that process, reactive oxygen species are generated, which in turn exert apoptotic events on the cancer cells. Objectives: We evaluated the cytotoxic effects of UV-irradiated mTiNPs on prostate cancer (PCa) cell line PC3 with the aim of demonstrating that the interaction between UV-light and mTiNPs positively impacts the nanomaterial’s cytotoxic efficiency. Moreover, we assessed the differential expression of key oncomiRs and tumor suppressor (TS) miRNAs, as well as their associated target genes, in cells undergoing this treatment. Methods: PBS-suspended mTiNPs exposed to 290 nm UV light were added at different concentrations to PC3 cells. Cell viability was determined after 24 h with a crystal violet assay. Then, the obtained IC50 concentration of UV-nanomaterial was applied to a new PC3 cell culture, and the expression of a set of miRNAs and selected target genes was evaluated via qRT-PCR. Results: The cells exposed to photo-activated mTiNPs required 4.38 times less concentration of the nanomaterial than the group exposed to non-irradiated mTiNPs to achieve the half-maximal inhibition, demonstrating an improved cytotoxic performance of the UV-irradiated mTiNPs. Moreover, the expression of miR-18a-5p, miR-21-5p, and miR-221-5p was downregulated after the application of UV-mTiNPs, while TS miR-200a-5p and miR-200b-5p displayed an upregulated expression. Among the miRNA target genes, PTEN was found to be upregulated after the treatment, while BCL-2 and TP53 were underexpressed. Conclusions: Our cytotoxic outcomes coincided with previous reports performed in other cancer cell lines, strongly suggesting UV-irradiated mTiNPs as a promising nano-therapeutic approach against PCa. On the other hand, to the best of our knowledge, this is the first report exploring the impact of UV-irradiated mTiNPs on key onco- and TS microRNAs in PCa cells.

Zanthoxylum piperitum Benn. Attenuates Monosodium Urate-Induced Gouty Arthritis: A Network Pharmacology Investigation of Its Anti-Inflammatory Mechanisms.

Background: Monosodium urate crystal accumulation in the joints is the cause of gout, an inflammatory arthritis that is initiated by elevated serum uric acid levels. It is the most prevalent form of inflammatory arthritis, affecting millions worldwide, and requires effective treatments. The necessity for alternatives with fewer side effects is underscored by the frequent adverse effects of conventional therapies, such as urate-lowering drugs. IL-1β is a potential therapeutic target due to its significant role in the inflammatory response induced by MSU. Zanthoxylum piperitum Benn. (ZP), a shrub that possesses antibacterial, antioxidant, and anti-inflammatory properties, has demonstrated potential in the treatment of inflammatory conditions. Methods: For anti-inflammatory properties of ZP, Raw264.7 cell stimulated LPS were treated ZP and using RNA-seq with Bone marrow derived macrophage, we observed to change inflammatory gene. Pharmacological networks were conducted to select target gene associated with ZP. For in vivo, mice were injected MSU in footpad for induce gouty arthritis model. The components of ZP were analyzed using GC-MS, and distilled extracts of ZP (deZP) were prepared. Results: In vitro, deZP decreased inflammatory cytokines. However, in vivo, it also decreased paw thickness and IL-1β levels. The anti-inflammatory effects of deZP are believed to be mediated through the NLRP3 inflammasome pathway, as indicated by RNA sequencing and network pharmacology analyses. Conclusions: ZP has an anti-inflammatory effect and regulation of the NLRP3 inflammasome in vitro and in vivo. Further research, including clinical trials, is required to confirm the safety of deZP, determine the optimal dosing, and evaluate its long-term effects.

Exploring the transcriptomic and m6a landscape of human chromosome 17 in breast cancer: A combined RNA-seq and MeRIP-Seq analysis

Abstract. N6-methyladenosine (m6A), recognized as the most prevalent post-transcriptional modification in organisms, has been substantiated to exert a significant influence on the genetic mechanisms underlying breast cancer. To delve deeper into the disparities in expression, modification, and interactions between mRNA and m6A in breast cancer (BC), we conducted RNA-seq and MeRIP-Seq analyses on eight human breast tissue transcriptome samples acquired from the GEO database. combining two single omics and integrated analysis, the study unveiled the mRNA and m6A expression patterns, differential genes, and enriched pathway functional disparities in BC (on Chromosome 17): (1) A confidence list comprised of 53 differential genes was derived by intersecting the 72 identified differentially expressed mRNA genes and the 781 differentially modified m6A genes; (2) The three-way analysis revealed two distinct types of pathways related to BC: ABC transporter activity and Response to epidermal growth factor. Among these, the EGF pathways exhibited the closest association with the differential m6A modifications. Meanwhile, all three BC subtypes enriched in DisGeNET were all linked to EGF; (3) By integrating PPI and enrichment analysis, we selected target genes from the confidence gene list, which included ABCA6, ABCA8, and ABCA10, known to be involved in regulating ABC transporters, and ERBB2, a central hub gene in PPI with a pivotal role in the EGF pathway and Her2-positive BC subtype under m6A differential modification. Subsequent research may uncover RNA-binding proteins for these target genes and offer effective drug design targets for BC.

Physiological and pathological roles of the transcriptional kinases CDK12 and CDK13 in the central nervous system.

The cyclin-dependent kinases 12 (CDK12) and 13 (CDK13) govern several steps of gene expression, including transcription, RNA processing and translation. The main target of CDK12/13 is the serine 2 residue of the carboxy-terminal domain of RNA polymerase II (RNAPII), thus influencing the directionality, elongation rate and processivity of the enzyme. The CDK12/13-dependent regulation of RNAPII activity influences the expression of selected target genes with important functional roles in the proliferation and viability of all eukaryotic cells. Neuronal cells are particularly affected by the loss of CDK12/13, as result of the high dependency of neuronal genes on RNAPII processivity for their expression. Deregulation of CDK12/13 activity strongly affects brain physiology by influencingthe stemness potential and differentiation properties of neuronal precursor cells. Moreover, mounting evidence also suggest the involvement of CDK12/13 in brain tumours. Herein, we discuss the functional role(s) of CDK12 and CDK13 in gene expression regulation and highlight similarities and differences between these highly homologous kinases, with particular attention to their impact on brain physiology and pathology. Lastly, we provide an overview of CDK12/13 inhibitors and of their efficacy in brain tumours and other neoplastic diseases.

Genome-wide selection of potential target candidates for RNAi against Nilaparvata lugens

BackgroundNilaparvata lugens is one of the most destructive pests of rice. RNAi-based N. lugens control offers one alternative strategy to traditional chemical insecticides. However, selection of potential target for RNAi against N. lugens remains a major challenge. Only two target genes for nuclear transgenic N. lugens-resistant plants have been screened. Importantly, only one or few potential target genes against N. lugens were screened every time by knowledge of essential genes from model organisms in previous study.ResultsHere, in silico genome-wide selection of potential target genes against N. lugens through homology comparison was performed. Through genome synteny comparisons, about 3.5% of Drosophila melanogaster genome was found to have conserved genomic synteny with N. lugens genome. By using N. lugens proteins to search D. melanogaster homologs defining lethal or sterile phenotype, 358 N. lugens genes were first screened as putative target genes. Transgenic rice lines expressing dsRNA of randomly selected gene (NlRan or NlSRP54) from 358 putative target genes enhanced resistance to N. lugens. After expression check and safety check, 115 N. lugens genes were screened as potential target candidates.ConclusionThe combined efforts in this study firstly provide one in silico genome-wide homology-based screening approach for RNAi-based target genes against N. lugens, which not only offer one new opportunity to batch select potential target candidates in pests of interest, but also will facilitate the selection of RNAi target in many pest species by providing more than one hundred potential target candidates.

Pancancer analysis of the interactions between CTNNB1 and infiltrating immune cell populations.

Recently, evidence has indicated that CTNNB1 is important in a variety of malignancies. However, how CTNNB1 interacts with immune cell infiltration remains to be further investigated. In this study, we focused on the correlations between CTNNB1 and tumorigenesis, tumor progression, mutation, phosphorylation, and prognosis via gene expression profiling interaction analysis; TIMER 2.0, cBioPortal, GTEx, CPTAC, and GEPIA2 database analyses; and R software. CTNNB1 mutations are most found in uterine endometrioid carcinoma and hepatocellular carcinoma. However, no CTNNB1 mutations were found to be associated with a poor prognosis. In addition, CTNNB1 DNA methylation levels were higher in normal tissues than in tumor tissues in cancer except for breast invasive carcinoma, which had higher methylation levels in tumor tissues. The phosphorylation level of the S675 and S191 sites of CTNNB1 was greater in the primary tumor tissues in the clear cell renal cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, and breast cancer datasets but not in the glioblastoma multiform dataset. As for, with respect to immune infiltration, CD8 + T-cell infiltration was negatively correlated with the expression of CTNNB1 in thymoma and uterine corpus endometrial carcinoma. The CTNNB1 level was found to be positively associated with the infiltration index of the corresponding fibroblasts in the TCGA tumors of colon adenocarcinoma, human papillomavirus-negative head and neck squamous cell carcinoma, mesothelioma, testicular germ cell tumor, and thymoma. We also identified the top CTNNB1-correlated genes in the TCGA projects and analyzed the expression correlation between CTNNB1 and selected target genes, including PPP4R2, RHOA, and SPRED1. Additionally, pathway enrichment suggested that NUMB is involved in the Wnt pathway. This study highlights the predictive role of CTNNB1 across cancers, suggesting that CTNNB1 might serve as a potential biomarker for the diagnosis and prognosis evaluation of various malignant tumors.

Non-electrophilic NRF2 activators promote wound healing in human keratinocytes and diabetic mice and demonstrate selective downstream gene targeting

The transcription factor NRF2 plays an important role in many biological processes and is a promising therapeutic target for many disease states. NRF2 is highly expressed in the skin and is known to play a critical role in diabetic wound healing, a serious disease process for which treatment options are limited. However, many existing NRF2 activators display off-target effects due to their electrophilic mechanism, underscoring the need for alternative approaches. In this work, we investigated two recently described non-electrophilic NRF2 activators, ADJ-310 and PRL-295, and demonstrated their efficacy in vitro and in vivo in human keratinocytes and Leprdb/db diabetic mice. We also compared the downstream targets of PRL-295 to those of the widely used electrophilic NRF2 activator CDDO-Me by RNA sequencing. Both ADJ-310 and PRL-295 maintained human keratinocyte cell viability at increasing concentrations and maintained or improved cell proliferation over time. Both compounds also increased cell migration, improving in vitro wound closure. ADJ-310 and PRL-295 enhanced the oxidative stress response in vitro, and RNA-sequencing data showed that PRL-295 activated NRF2 with a narrower transcriptomic effect than CDDO-Me. In vivo, both ADJ-310 and PRL-295 improved wound healing in Leprdb/db diabetic mice and upregulated known downstream NRF2 target genes in treated tissue. These results highlight the non-electrophilic compounds ADJ-310 and PRL-295 as effective, innovative tools for investigating the function of NRF2. These compounds directly address the need for alternative NRF2 activators and offer a new approach to studying the role of NRF2 in human disease and its potential as a therapeutic across multiple disease states.

CRISPR-GEM: A Novel Machine Learning Model for CRISPR Genetic Target Discovery and Evaluation.

CRISPR gene editing strategies are shaping cell therapies through precise and tunable control over gene expression. However, limitations in safely delivering high quantities of CRISPR machinery demand careful target gene selection to achieve reliable therapeutic effects. Informed target gene selection requires a thorough understanding of the involvement of target genes in gene regulatory networks (GRNs) and thus their impact on cell phenotype. Effective decoding of these complex networks has been achieved using machine learning models, but current techniques are limited to single cell types and focus mainly on transcription factors, limiting their applicability to CRISPR strategies. To address this, we present CRISPR-GEM, a multilayer perceptron (MLP) based synthetic GRN constructed to accurately predict the downstream effects of CRISPR gene editing. First, input and output nodes are identified as differentially expressed genes between defined experimental and target cell/tissue types, respectively. Then, MLP training learns regulatory relationships in a black-box approach allowing accurate prediction of output gene expression using only input gene expression. Finally, CRISPR-mimetic perturbations are made to each input gene individually, and the resulting model predictions are compared to those for the target group to score and assess each input gene as a CRISPR candidate. The top scoring genes provided by CRISPR-GEM therefore best modulate experimental group GRNs to motivate transcriptomic shifts toward a target group phenotype. This machine learning model is the first of its kind for predicting optimal CRISPR target genes and serves as a powerful tool for enhanced CRISPR strategies across a range of cell therapies.

A Comparison of GAPDH and ACTB As Internal Control for Gene Expression Studies in Different Cancer Cell Lines

Housekeeping genes are used as internal controls to normalize the expression of target genes in gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Actin beta (ACTB) are frequently preferred as housekeeping genes in gene expression studies. Due to the general alterations in the gene expression pattern in cancer cases, the selection of the appropriate housekeeping genes for these studies are challenging. In this study, we aimed to analyze the expression of the well-known housekeeping genes GAPDH and ACTB in 6 different cancer cell lines. Furthermore, the relative gene expression of the selected target gene (Tissue inhibitor of metalloproteinases 2-TIMP2) was normalized separately using GAPDH and ACTB and the obtained results were compared with each other. Finally, the stability of GAPDH and ACTB was analyzed using the in-silico tool, Bestkeeper. As a result of the study, it is found that the expression of GAPDH and ACTB were significantly different in the Jurkat (p< 0.01), MOLT4 (p< 0.05), REH (p< 0.001) and HT29 (p< 0.001) cell lines. Based on this finding, significantly different relative target gene expression values were obtained in different cell lines depending on whether the selected housekeeping gene was GAPDH or ACTB. In addition, GAPDH was found to show less variation among the samples used in all cell lines and more stability based on Bestkeeper analysis. These results support that the appropriate housekeeping gene selection, especially in cancer cell lines, may be an effective factor in obtaining accurate results for the studies in the field provide guidance to researchers. Keywords: Cancer cell lines, Gene expression, Housekeeping genes, GAPDH, ACTB

Mediterranean Diet Modulation of Neuroinflammation-Related Genes in Elderly Adults at High Cardiovascular Risk.

Individuals with dementia and neurodegenerative diseases (NDDs) often suffer from cardiovascular diseases (CVDs). Neuroinflammation driven by conditions involved in CVDs is linked to disruptions in the central nervous system triggering immune reactions, perpetuating an "inflammatory-like" environment. The Mediterranean diet (MedDiet), known for its anti-inflammatory and antioxidant properties, has been proposed as a key factor to attenuate these risks. Blood nuclear cell samples were collected from 134 participants of the PREDIMED trial, which randomized participants to three diets: one supplemented with extra-virgin olive oil (MedDiet-EVOO), another with nuts (MedDiet-Nuts), and a low-fat control diet. These samples were analyzed at baseline and 12-month follow-up to assess the impact of these dietary interventions on gene expression markers. We first selected target genes by analyzing intersections between NDD and CVD associations. Significant gene expression changes from baseline to 12 months were observed in the participants allocated to the MedDiet-EVOO, particularly in CDKN2A, IFNG, NLRP3, PIK3CB, and TGFB2. Additionally, TGFB2 expression changed over time in the MedDiet-Nuts group. Comparative analyses showed significant differences in TGFB2 between MedDiet-EVOO and control, and in NAMPT between MedDiet-Nuts and control. Longitudinal models adjusted for different covariates also revealed significant effects for TGFB2 and NAMPT. In conclusion, our results suggest that one year of traditional MedDiet, especially MedDiet-EVOO, modulates gene expression associated with CVD risk and NDDs in older adults at high CV risk.

Comparative transcriptomic insights into key genes for biomass production and lipid synthesis in Chlorella sorokiniana mutated by argon and air atmospheric and room temperature plasma at different culture stages: Common mechanisms and unique differences

Comparative transcriptomic insights into key genes for biomass production and lipid synthesis in Chlorella sorokiniana mutated by argon and air atmospheric and room temperature plasma at different culture stages: Common mechanisms and unique differences

Curcumin suppresses cell viability in breast cancer cell line by affecting the expression of miR-15a-5p

Abstract Objectives Curcumin plays a leading role as an epigenetic regulator in cancer. miR-15a-5p is a crucial non-coding RNA for breast cancer (BRCA) and various cancers due to its tumor suppressor role. In our study, we aimed to examine the curcumin/miR-15a-5p/target gene interaction in BRCA cells. Methods The effects of curcumin and miR-15a-5p on cell viability in the MCF7 cells were examined using the WST8 technique. The cell migration was determined using scratch wound assay. miR-15a-5p level was detected in curcumin-treated cells and miR-15a-5p transfected cells compared to control groups by RT-qPCR. Overexpressed genes in BRCA were found by bioinformatics tools (GSE41970 and TCGA). miR15a-5p potential target genes in the miRNet tool were selected in overlapped genes between GSE41970 and TCGA. Survival analysis of the selected genes was examined using the GEPIA2 tool. Relative expression levels of four selected genes were examined via qPCR. Results Cell viability and scratch-wound closure rate were reduced in curcumin-treated and miR-15a-5p mimic transfected MCF7 cells. miR15a-5p overexpressed in curcumin-treated and miR-15a-5p transfected cells. Eighty-three dysregulated upregulated genes were detected (in GSE41970 and TCGA). Among the possible target genes of miR-15a-5p in the miRNet tool, 10 upregulated genes were detected overlapping with GSE41970 and TCGA. CCNE1 and CHEK1 genes were found to be important for survival in BRCA. CCNE1 and BMI1 were decreased in curcumin-treated and miR-15a-5p transfected cells. Conclusions Curcumin treatment increased miR-15a-5p and downregulated selected target genes. Curcumin/miR-15a-5p interaction may be a much stronger negative regulator of the CCNE1 and BMI1 genes in BRCA.

Genomics of natural populations: gene conversion events reveal selected genes within the inversions of Drosophila pseudoobscura.

When adaptive phenotypic variation or quantitative trait loci map within an inverted segment of a chromosome, researchers often despair because the suppression of crossing over will prevent the discovery of selective target genes that established the rearrangement. If an inversion polymorphism is old enough, then the accumulation of gene conversion tracts offers the promise that quantitative trait loci or selected loci within inversions can be mapped. The inversion polymorphism of Drosophila pseudoobscura is a model system to show that gene conversion analysis is a useful tool for mapping selected loci within inversions. D. pseudoobscura has over 30 different chromosomal arrangements on the third chromosome (Muller C) in natural populations and their frequencies vary with changes in environmental habitats. Statistical tests of five D. pseudoobscura gene arrangements identified outlier genes within inverted regions that had potentially heritable variation, either fixed amino acid differences or differential expression patterns. We use genome sequences of the inverted third chromosome (Muller C) to infer 98,443 gene conversion tracts for a total coverage of 142 Mb or 7.2× coverage of the 19.7 Mb chromosome. We estimated gene conversion tract coverage in the 2,668 genes on Muller C and tested whether gene conversion coverage was similar among arrangements for outlier vs non-outlier loci. Outlier genes had lower gene conversion tract coverage among arrangements than the non-outlier genes suggesting that selection removes exchanged DNA in the outlier genes. These data support the hypothesis that the third chromosome in D. pseudoobscura captured locally adapted combinations of alleles prior to inversion mutation events.

Protein aggregation in plant mitochondria lacking Lon1 inhibits translation and induces unfolded protein responses.

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

Rapid Identification of the Spruce Bark Beetle <i>Ips typographus</i> (Linnaeus) Basing on a New Amplification and Analysis Platform

Insects, one of the major disturbance agents, are regarded as a big challenge to forests. Bark beetles (Coleoptera: Curculionidae: Scolytinae) are among the most destructive pests around the world. The European spruce bark beetle <i>I. typographus </i>(Linnaeus) is considered the most dangerous species to mature spruce forests throughout Eurasia. In order to improve efficiency, accuracy, and operability of identification, a rapid, simple, highly sensitive and specific screening method is in urgent need. In this study, a rapid classification approach for <i>I. typographus</i> was established based on the enzyme-mediated duplex exponential amplification (EmDEA) amplification and analysis platform. The method development process consists of target gene selection, primer design, primer screening, and method validation. Parameter analysis demonstrated that this new method has a detection limit of 1.96×103 copies/μL, which is comparable to conventional molecular tools such as PCR. Stable repeatability and high specificity were confirmed by testing 5 samples of <i>I. typographus</i> and 4 related beetles. Besides, this screening protocol was easy to use, and could be completed in 30 min. With the advantage of isothermal amplification, this method could be further applied in non-laboratory scenarios such as port rapid screening and wild survey. This rapid screening method for <i>I. typographus</i> is believed to assist precise prediction and efficient prevention of exotic insect species.

CRISPR-GEM: A Novel Machine Learning Model for CRISPR Genetic Target Discovery and Evaluation.

CRISPR gene editing strategies are shaping cell therapies through precise and tunable control over gene expression. However, achieving reliable therapeutic effects with improved safety and efficacy requires informed target gene selection. This depends on a thorough understanding of the involvement of target genes in gene regulatory networks (GRNs) that regulate cell phenotype and function. Machine learning models have been previously used for GRN reconstruction using RNA-seq data, but current techniques are limited to single cell types and focus mainly on transcription factors. This restriction overlooks many potential CRISPR target genes, such as those encoding extracellular matrix components, growth factors, and signaling molecules, thus limiting the applicability of these models for CRISPR strategies. To address these limitations, we have developed CRISPR-GEM, a multi-layer perceptron (MLP)-based synthetic GRN constructed to accurately predict the downstream effects of CRISPR gene editing. First, input and output nodes are identified as differentially expressed genes between defined experimental and target cell/tissue types respectively. Then, MLP training learns regulatory relationships in a black-box approach allowing accurate prediction of output gene expression using only input gene expression. Finally, CRISPR-mimetic perturbations are made to each input gene individually and the resulting model predictions are compared to those for the target group to score and assess each input gene as a CRISPR candidate. The top scoring genes provided by CRISPR-GEM therefore best modulate experimental group GRNs to motivate transcriptomic shifts towards a target group phenotype. This machine learning model is the first of its kind for predicting optimal CRISPR target genes and serves as a powerful tool for enhanced CRISPR strategies across a range of cell therapies.

Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical cancer cell line.

Introduction: Cervical cancer is one of the leading causes of death among women globally due to the limitation of current treatment methods and their associated adverse side effects. Launaea cornuta is used as traditional medicine for the treatment of a variety of diseases including cancer. However, there is no scientific validation on the antiproliferative activity of L. cornuta against cervical cancer. Objective: This study aimed to evaluate the selective antiproliferative, cytotoxic and antimigratory effects of L. cornuta and to explore its therapeutical mechanisms in human cervical cancer cell lines (HeLa-229) through a network analysis approach. Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate fraction on the proliferation of cervical cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay and the antimigratory effect was assessed by wound healing assays. Compounds were analysed using the qualitative colour method and gas chromatography-mass spectroscopy (GC-MS). Subsequently, bioinformatic analyses, including the protein-protein interaction (PPI) network analysis, Gene Ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, were performed to screen for potential anticervical cancer therapeutic target genes of L. cornuta. Molecular docking (MD) was performed to predict and understand the molecular interactions of the ligands against cervical cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the network analysis results. Results: L. cornuta ethyl acetate fraction exhibited a remarkable cytotoxic effect on HeLa-229 proliferation (IC50 of 20.56 ± 2.83μg/mL) with a selectivity index (SI) of 2.36 with minimal cytotoxicity on non-cancerous cells (Vero-CCL 81 (IC50 of 48.83 ± 23.02). The preliminary screening revealed the presence of glycosides, phenols, saponins, terpenoids, quinones, and tannins. Thirteen compounds were also identified by GC-MS analysis. 124 potential target genes associated with the effect of L. cornuta ethyl acetate fraction on human cervical cancer were obtained, including AKT1, MDM2, CDK2, MCL1 and MTOR were identified among the top hub genes and PI3K/Akt1, Ras/MAPK, FoxO and EGFR signalling pathways were identified as the significantly enriched pathways. Molecular docking results showed that stigmasteryl methyl ether had a good binding affinity against CDK2, ATK1, BCL2, MDM2, and Casp9, with binding energy ranging from -7.0 to -12.6kcal/mol. Tremulone showed a good binding affinity against TP53 and P21 with -7.0 and -8.0kcal/mol, respectively. This suggests a stable molecular interaction of the ethyl acetate fraction of L. cornuta compounds with the selected target genes for cervical cancer. Furthermore, RT-qPCR analysis revealed that CDK2, MDM2 and BCL2 were downregulated, and Casp9 and P21 were upregulated in HeLa-229 cells treated with L. cornuta compared to the negative control (DMSO 0.2%). Conclusion: The findings indicate that L. cornuta ethyl acetate fraction phytochemicals modulates various molecular targets and pathways to exhibit selective antiproliferative and cytotoxic effects against HeLa-229 cells. This study lays a foundation for further research to develop innovative clinical anticervical cancer agents.

Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

The use of cell and larval assays to identify target genes for RNA interference-meditated control of the Australian sheep blowfly (Lucilia cuprina).

Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.