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17 pages, 1675 KiB  
Article
Gene Copy Number Dictates Extracellular Vesicle Cargo
by Sumeet Poudel, Zhiyong He, Jerilyn Izac and Lili Wang
Int. J. Mol. Sci. 2025, 26(12), 5496; https://doi.org/10.3390/ijms26125496 - 8 Jun 2025
Viewed by 674
Abstract
Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell [...] Read more.
Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell communication. However, the way in which the genome signature regulates EV cargo has not been well studied. This study aimed to understand how genetics impact EV cargo loading. EVs were isolated from vector copy number cells with a fluorescent reporter (GFP) with varying inserted transgene copies and from NIST SRM 2373 cells (MDA-MB-231, MDA-MB-453, SK-BR-3, and BT-474), which contain varying copies of the HER2 gene. Spectradyne nCS1 was utilized to count EVs and measure size distribution. Imaging Flow Cytometry was used to analyze the surface protein content of single EVs and for total EV counts. The RNA content of the EVs was measured using ddPCR. Our results from stable reporter cell lines and breast cancer cell lines suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of a reporter gene (GFP) or a naturally occurring gene (HER2) from breast cancer cells correlated with increasing EV counts positive for the protein cargo compared to total EV counts until a copy threshold was reached. This study has broad implications for understanding EV biology in the context of cancer biology, diagnostics, EV biology/manufacturing, and therapeutic delivery. Full article
(This article belongs to the Section Molecular Biology)
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17 pages, 3653 KiB  
Article
Genome-Wide Identification and Characterization of the mTERF Gene Family in Spinach and the Role of SomTERF5 in Response to Heat Stress
by Ziyue Sun, Li Li, Yaqi Liu, Yanshuang Liu, Gaojian Li, Yueyue Li, Qingbo Yu, Meihong Sun and Xiaofeng Xu
Plants 2025, 14(11), 1570; https://doi.org/10.3390/plants14111570 - 22 May 2025
Viewed by 477
Abstract
Spinach (Spinacia oleracea L.), a globally consumed, nutrient-dense vegetable, contains diverse vitamins and minerals. However, elevated temperatures can constrain yield by interrupting leaf development and photosynthetic efficiency. The mitochondrial transcription termination factor (mTERF) family, which regulates organellar gene expression, plays crucial roles [...] Read more.
Spinach (Spinacia oleracea L.), a globally consumed, nutrient-dense vegetable, contains diverse vitamins and minerals. However, elevated temperatures can constrain yield by interrupting leaf development and photosynthetic efficiency. The mitochondrial transcription termination factor (mTERF) family, which regulates organellar gene expression, plays crucial roles in plant growth and photosynthetic regulation. Thus, characterization of the spinach mTERF (SomTERF) family is critical for elucidating thermotolerance mechanisms in this crop. In this study, we systematically identified 31 SomTERF genes from the spinach genome, which are distributed across five chromosomes and nine unassembled genomic scaffolds. Subcellular localization predictions indicated that these proteins predominantly target chloroplasts and mitochondria. Conserved domain analyses confirmed that all SomTERF proteins possess canonical mTERF domains and ten conserved motifs. Phylogenetic clustering segregated these proteins into nine distinct subgroups (I–IX), with significant divergence observed in gene copy numbers among subgroups. Cis-element screening identified an abundance of heat-, cold-, and hormone-responsive motifs within SomTERF promoter regions. Notably, seven members (including SomTERF5) exhibited pronounced enrichment of heat shock elements (HSEs). Organ-specific expression profiling revealed preferential leaf expression of these seven genes. Comparative RT-qPCR in heat-sensitive (Sp73) and heat-tolerant (Sp75) cultivars under thermal stress demonstrated genotype-dependent expression dynamics. Functional validation of SomTERF5 was achieved through cloning, and transgenic Arabidopsis overexpressing SomTERF5 showed significantly enhanced thermotolerance, as evidenced by improved survival rates following heat treatment. Yeast two-hybrid (Y2H) assays further revealed physical interaction between SomTERF5 and SopTAC2. This study provides a comprehensive foundation for understanding mTERF-mediated developmental regulation and advanced molecular breeding strategies for developing heat-resilient spinach varieties. Full article
(This article belongs to the Special Issue Growth, Development, and Stress Response of Horticulture Plants)
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13 pages, 2630 KiB  
Article
Rapid and Economic Baculovirus Titer Determination Using a Novel Transgenic Sf9-QE Cell Line
by Hyuk-Jin Moon, Hyun-Jung Kim, Dong-Hyun Lee, Seo-Yeong Mun and Soo-Dong Woo
Insects 2025, 16(4), 426; https://doi.org/10.3390/insects16040426 - 17 Apr 2025
Viewed by 847
Abstract
A baculovirus expression system (BES) for the production of recombinant proteins requires rapid and easy virus titer determination. In this study, a novel direct titration method was developed using a novel Sf9-QE cell line to easily and economically determine virus titers in a [...] Read more.
A baculovirus expression system (BES) for the production of recombinant proteins requires rapid and easy virus titer determination. In this study, a novel direct titration method was developed using a novel Sf9-QE cell line to easily and economically determine virus titers in a short time. This direct titration method can determine virus titers by directly counting the initially infected cells. This method requires the rapid identification of the initial virus-infected cells. The genome of Sf9-QE cells, which fluoresce upon virus infection, was found to contain at least seven copy numbers of the enhanced green fluorescent protein (EGFP) transgene. This result suggests that Sf9-QE cells in the early stages of virus infection can be identified by the high expression of EGFP. It was also shown that for accurate virus titration using the direct titration method, Sf9-QE cells should be used within 3 d of subculturing. Additionally, counting fluorescent cells to establish virus infection should be performed within 15 to 30 h after virus infection for reliable virus titration. The direct titration method using Sf9-QE cells provides a rapid, reliable, and cost-effective alternative for determining baculovirus titers in BES research. Full article
(This article belongs to the Special Issue Research on Insect Molecular Biology)
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39 pages, 11200 KiB  
Article
Analysis of Hepatic Lentiviral Vector Transduction: Implications for Preclinical Studies and Clinical Gene Therapy Protocols
by Peirong Hu, Yajing Hao, Wei Tang, Graham H. Diering, Fei Zou and Tal Kafri
Viruses 2025, 17(2), 276; https://doi.org/10.3390/v17020276 - 17 Feb 2025
Viewed by 1396
Abstract
Lentiviral vector-transduced T cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the effects of host genetic variation on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge gap, we characterized [...] Read more.
Lentiviral vector-transduced T cells were approved by the FDA as gene therapy anti-cancer medications. Little is known about the effects of host genetic variation on the safety and efficacy of the lentiviral vector gene delivery system. To narrow this knowledge gap, we characterized hepatic gene delivery by lentiviral vectors across the Collaborative Cross (CC) mouse genetic reference population. For 24 weeks, we periodically measured hepatic luciferase expression from lentiviral vectors in 41 CC mouse strains. Hepatic and splenic vector copy numbers were determined. We report that the CC mouse strains showed highly diverse outcomes following lentiviral gene delivery. For the first time, a moderate correlation between mouse-strain-specific sleeping patterns and transduction efficiency was observed. We associated two quantitative trait loci (QTLs) with intrastrain variations in transduction phenotypes, which mechanistically relates to the phenomenon of metastable epialleles. An additional QTL was associated with the kinetics of hepatic transgene expression. Genes found in the above QTLs are potential targets for personalized gene therapy protocols. Importantly, we identified two mouse strains that open new directions for characterizing continuous viral vector silencing and HIV latency. Our findings suggest that wide-range patient-specific outcomes of viral vector-based gene therapy should be expected. Thus, novel clinical protocols should be considered for non-fatal diseases. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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26 pages, 3161 KiB  
Article
Levels of Amyloid Beta () Expression in the Caenorhabditis elegans Neurons Influence the Onset and Severity of Neuronally Mediated Phenotypes
by Neha Sirwani, Shannon M. Hedtke, Kirsten Grant, Gawain McColl and Warwick N. Grant
Cells 2024, 13(18), 1598; https://doi.org/10.3390/cells13181598 - 23 Sep 2024
Cited by 1 | Viewed by 1915
Abstract
A characteristic feature of Alzheimer’s disease (AD) is the formation of neuronal extracellular senile plaques composed of aggregates of fibrillar amyloid β () peptides, with the Aβ1-42 peptide being the most abundant species. These peptides have been proposed to contribute [...] Read more.
A characteristic feature of Alzheimer’s disease (AD) is the formation of neuronal extracellular senile plaques composed of aggregates of fibrillar amyloid β () peptides, with the Aβ1-42 peptide being the most abundant species. These peptides have been proposed to contribute to the pathophysiology of the disease; however, there are few tools available to test this hypothesis directly. In particular, there are no data that establish a dose–response relationship between peptide expression level and disease. We have generated a panel of transgenic Caenorhabditis elegans strains expressing the human Aβ1-42 peptide under the control of promoter regions of two pan-neuronal expressed genes, snb-1 and rgef-1. Phenotypic data show strong age-related defects in motility, subtle changes in chemotaxis, reduced median and maximum lifespan, changes in health span indicators, and impaired learning. The Aβ1-42 expression level of these strains differed as a function of promoter identity and transgene copy number, and the timing and severity of phenotypes mediated by Aβ1-42 were strongly positively correlated with expression level. The pan-neuronal expression of varying levels of human Aβ1-42 in a nematode model provides a new tool to investigate the in vivo toxicity of neuronal expression and the molecular and cellular mechanisms underlying AD progression in the absence of endogenous peptides. More importantly, it allows direct quantitative testing of the dose–response relationship between neuronal peptide expression and disease for the first time. These strains may also be used to develop screens for novel therapeutics to treat Alzheimer’s disease. Full article
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17 pages, 1303 KiB  
Article
A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications
by Gertrud Wiedemann, Ulrike Bacher, Raphael Joncourt, Françoise Solly, Corinne C. Widmer, Sacha Zeerleder, Urban Novak, Thomas Pabst and Naomi A. Porret
Int. J. Mol. Sci. 2024, 25(16), 8556; https://doi.org/10.3390/ijms25168556 - 6 Aug 2024
Cited by 1 | Viewed by 2494
Abstract
In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed [...] Read more.
In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies. Full article
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19 pages, 5366 KiB  
Article
Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line
by Shengbo Zhao, Junling Luo, Min Tang, Chi Zhang, Miaoying Song, Gang Wu and Xiaohong Yan
Int. J. Mol. Sci. 2024, 25(1), 174; https://doi.org/10.3390/ijms25010174 - 22 Dec 2023
Viewed by 1593
Abstract
The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was [...] Read more.
The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was screened from the Brassica napus (B. napus) RNAi library. Three different methods confirmed that eight copies of T-DNA are present in the P198 genome. However, only four insertion positions were identified in three chromosomes using fusion primer and nested integrated polymerase chain reaction. Therefore, the T-DNA insertion sites and copy number were further investigated using Oxford Nanopore Technologies (ONT) sequencing, and it was found that at least seven copies of T-DNA were inserted into three insertion sites. Based on the obtained T-DNA insertion sites and hairpin RNA (hpRNA) cassette sequences, three candidate genes related to the P198 phenotype were identified. Furthermore, the potential differentially expressed genes and pathways involved in the dwarfism and sterility phenotype of P198 were investigated by RNA-seq. These results demonstrate the advantage of applying ONT sequencing to investigate the molecular characteristics of transgenic lines and expand our understanding of the complex molecular mechanism of dwarfism and male sterility in B. napus. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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11 pages, 18959 KiB  
Article
Downregulation of the INDEHISCENT Gene by RNAi Resulted in Desired Pod Shatter Reduction of Lepidium campestre in Subsequent Generations
by Emelie Ivarson, Annelie Ahlman, Jan-Eric Englund, Ida Lager and Li-Hua Zhu
Int. J. Mol. Sci. 2023, 24(21), 15943; https://doi.org/10.3390/ijms242115943 - 3 Nov 2023
Cited by 1 | Viewed by 1495
Abstract
Wild species field cress (Lepidium campestre) has favorable agronomic traits, making it a good candidate for future development as an oil and catch crop. However, the species is very prone to pod shatter, resulting in severe yield losses. This is one [...] Read more.
Wild species field cress (Lepidium campestre) has favorable agronomic traits, making it a good candidate for future development as an oil and catch crop. However, the species is very prone to pod shatter, resulting in severe yield losses. This is one of the important agronomic traits that needs to be improved in order to make this species economically viable. In this study, we cloned the L. campestre INDEHISCENT (LcIND) gene and prepared two LcIND-RNAi constructs with the IND promoter (long 400 bp and short 200 bp) from Arabidopsis. A number of stable transgenic lines were developed and evaluated in terms of pod shatter resistance. The majority of the transgenic lines showed increased resistance to pod shatter compared to the wild type, and this resistance was maintained in four subsequent generations. The downregulation of the LcIND gene by RNAi in the transgenic lines was confirmed by qRT-PCR analysis on T3 lines. Southern blot analysis showed that most of the analyzed lines had a single-copy integration of the transgene, which is desirable for further use. Our results show that it is possible to generate stable transgenic lines with desirable pod shatter resistance by downregulating the LcIND gene using RNAi in field cress, and thus speeding up the domestication process of this wild species. Full article
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11 pages, 2641 KiB  
Communication
Characterization of Mammary Tumors Arising from MMTV-PyVT Transgenic Mice
by Chien-Liang Liu, Wen-Chien Huang, Shih-Ping Cheng, Ming-Jen Chen, Chi-Hsin Lin, Shao-Chiang Chang and Yuan-Ching Chang
Curr. Issues Mol. Biol. 2023, 45(6), 4518-4528; https://doi.org/10.3390/cimb45060286 - 24 May 2023
Cited by 2 | Viewed by 2892
Abstract
Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic [...] Read more.
Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research. Full article
(This article belongs to the Topic Animal Models of Human Disease)
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13 pages, 1849 KiB  
Article
Effect of Soybean Protein Concentrate Preparation on Copy Numbers and Structural Characteristics of DNA from Genetically Modified Soybean
by Yan Du, Fusheng Chen, Kunlun Liu and Chen Chen
Foods 2023, 12(10), 2031; https://doi.org/10.3390/foods12102031 - 17 May 2023
Viewed by 1805
Abstract
To regulate the degradation of transgenic DNA and lay theoretical foundations for the rational utilization of genetically modified (GM) products, variations in copy numbers and structural characteristics of DNA from GM soybean event GTS 40-3-2 during soybean protein concentrate (SPC) preparation were evaluated. [...] Read more.
To regulate the degradation of transgenic DNA and lay theoretical foundations for the rational utilization of genetically modified (GM) products, variations in copy numbers and structural characteristics of DNA from GM soybean event GTS 40-3-2 during soybean protein concentrate (SPC) preparation were evaluated. Results showed that defatting and the first ethanol extraction were key procedures inducing DNA degradation. After these two procedures, copy numbers of the lectin and cp4 epsps targets decreased by more than 4 × 108, occupying 36.88–49.30% of the total copy numbers from raw soybean. Atomic force microscopy images visually revealed the degradation of DNA that thinned and shortened during SPC preparation. Circular dichroism spectra suggested a lower helicity of DNA from defatted soybean kernel flour and a conformation transition of DNA from B-type to A-type after ethanol extraction. The fluorescence intensity of DNA decreased during SPC preparation, verifying the DNA damage along this preparation chain. Full article
(This article belongs to the Section Food Quality and Safety)
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16 pages, 10176 KiB  
Article
Enhanced Antioxidant and Anticancer Potential of Artemisia carvifolia Buch Transformed with rol A Gene
by Amna Naheed Khan and Erum Dilshad
Metabolites 2023, 13(3), 351; https://doi.org/10.3390/metabo13030351 - 27 Feb 2023
Cited by 9 | Viewed by 2495
Abstract
Secondary metabolites have been shown to possess a range of biological functions. Flavonoids, due to their ability to scavenge ROS, are famous antioxidants. The plants of Artemisia species are rich sources of flavonoids; however, the amount of these metabolites is less. In the [...] Read more.
Secondary metabolites have been shown to possess a range of biological functions. Flavonoids, due to their ability to scavenge ROS, are famous antioxidants. The plants of Artemisia species are rich sources of flavonoids; however, the amount of these metabolites is less. In the current study, the flavonoid content was detected and then enhanced by genetically modifying the Artemisia carvifolia Buch with Agrobacterium tumefaciens strain GV3101 carrying rol A gene. The transformation of rol A gene was confirmed with PCR and the gene copy number was confirmed by Southern blot analysis. The HPLC analysis revealed the presence of catechin (3.19 ug/mg DW) and geutisic acid (2.22 ug/mg DW) in transformed plants, unlike wild-type plants. In transformed plants, all detected flavonoids (vanillic acid, rutin, catechine, gallic acid, syringic acid, caffeic acid, coumaric acid, geutisic acid, ferulic acid, and cinnamic acid) were increased up to several folds. Real-time qPCR revealed the higher expression levels of the genes for flavonoid biosynthesis enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in plants transformed with rol A genes, as the expression levels were increased up to 9–20-fold and 2–6-fold, respectively. The rol A transgenic lines T3 and T5 carrying two copies of rol A gene, particularly showed higher expression of both PAL and CHS gene, with the highest expression in T3 line. The transgenic lines demonstrated an average increase of 1.4-fold in the total phenolic content and 1–2-fold in the total flavonoid content as compared to wild-type plants. Total antioxidant capacity and total reducing power were increased up to an average of 1–2-fold and 1.5–2-fold respectively, along with increased free radical scavenging ability. Furthermore, the rol A gene transgenics were found to have much greater cytotoxic capacity than the A. carvifolia wild-type plant against the MCF7, HeLA, and HePG2 cancer cell lines. Current findings show that the rol A gene effectively increases the flavonoid content of A. carvifolia Buch, boosting the plant’s capacity as an antioxidant and an anticancer. This is the first-ever report, demonstrating the genetic transformation of Artemisia carvifolia Buch with rol A gene. Full article
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12 pages, 936 KiB  
Article
Assessment of Efficacy and Mechanism of Resistance to Soil-Applied PPO Inhibitors in Amaranthus palmeri
by Gulab Rangani, Aimone Porri, Reiofeli A. Salas-Perez, Jens Lerchl, Srikanth Kumar Karaikal, Juan Camilo Velásquez and Nilda Roma-Burgos
Agronomy 2023, 13(2), 592; https://doi.org/10.3390/agronomy13020592 - 18 Feb 2023
Cited by 3 | Viewed by 2354
Abstract
Resistance to protoporphyrinogen oxidase (PPO) inhibitors in Palmer amaranth is a major concern, given the high selection pressure and increasing number of populations with reduced sensitivity to PPO herbicides in the US. We evaluated the effect of five soil-applied herbicides on Palmer amaranth [...] Read more.
Resistance to protoporphyrinogen oxidase (PPO) inhibitors in Palmer amaranth is a major concern, given the high selection pressure and increasing number of populations with reduced sensitivity to PPO herbicides in the US. We evaluated the effect of five soil-applied herbicides on Palmer amaranth (Amaranthus palmeri S. Wats.) populations collected in 2014 and 2015 in Arkansas, USA. Soil-applied saflufenacil, sulfentrazone, and flumioxazin reduced the seedling emergence 91–100%; however, fomesafen and oxyfluorfen showed reduced (63–90%) efficacy on some populations. Target-site mutation (TSM) is the major mechanism of resistance to PPO herbicides; therefore, six populations showing resistance to soil-applied fomesafen were selected for molecular investigations. A total of 81 survivors were genotyped for all known resistance-conferring mutations. A total of 64% and 36% survivors had single and double TSMs, respectively, with 69% of plants carrying TSM in both alleles of PPO2. Three survivors from two populations showed an additional copy of PPO2, whereas all other survivors had one copy. Expression analysis showed 3- to 6-fold upregulation of PPO2 in all plants from resistant populations tested. Transgenic overexpression of WT-ApPPO2 and dG210-Apppo2 in A. thaliana confirmed the reduced sensitivity to soil-applied fomesafen compared to the wild type. Collectively, PPO inhibitors applied pre-emergence are still effective in controlling populations resistant to foliar-applied PPO herbicides. Mechanically, elevated expression of resistant PPO2, alongside functional TSM, contribute to reduced sensitivity to soil-applied fomesafen. Full article
(This article belongs to the Special Issue Herbicides Toxicology and Weeds Herbicide-Resistant Mechanism)
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17 pages, 3087 KiB  
Article
Cajanus platycarpus Flavonoid 3′5′ Hydroxylase_2 (CpF3′5′H_2) Confers Resistance to Helicoverpa armigera by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco
by Shaily Tyagi, Maniraj Rathinam, Narasimham Dokka, Nidhee Chaudhary, Lakkakula Satish, Prasanta K. Dash, Ajit Kumar Shasany and Rohini Sreevathsa
Int. J. Mol. Sci. 2023, 24(2), 1755; https://doi.org/10.3390/ijms24021755 - 16 Jan 2023
Cited by 10 | Viewed by 2572
Abstract
Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3′5′ [...] Read more.
Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3′5′ hydroxylase_2 (F3′5′H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3′5′H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3′5′H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3′5′H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3′5′H_2. The relevance of CpF3′5′H_2 in H. armigera resistance was further validated in CpF3′5′H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3′5′H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3′5′H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs. Full article
(This article belongs to the Special Issue Plant-Insect Interactions 2022)
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15 pages, 2089 KiB  
Article
Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum
by Irena Bočkaj, Tosca E. I. Martini, Marlinde J. Smit, Inna Armandari, Bjorn Bakker, René Wardenaar, Tiny G. J. Meeuwsen-de Boer, Petra L. Bakker, Diana C. J. Spierings, Eelco W. Hoving, Victor Guryev, Floris Foijer and Sophia W. M. Bruggeman
Int. J. Mol. Sci. 2022, 23(17), 9852; https://doi.org/10.3390/ijms23179852 - 30 Aug 2022
Cited by 2 | Viewed by 2153
Abstract
Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This [...] Read more.
Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma. Full article
(This article belongs to the Special Issue Molecular Biology and Translational Aspects in CNS Tumors 2.0)
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15 pages, 2158 KiB  
Article
Ectopic Expression of the Rice Grain-Size-Affecting Gene GS5 in Maize Affects Kernel Size by Regulating Endosperm Starch Synthesis
by Guoqing Dong, Hanxian Xiong, Wanyong Zeng, Jinhua Li and Dengxiang Du
Genes 2022, 13(9), 1542; https://doi.org/10.3390/genes13091542 - 26 Aug 2022
Cited by 4 | Viewed by 2419
Abstract
Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and [...] Read more.
Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and grain grouting rate. In this study, based on the GS5 transgenic maize obtained through transgenic technology with specific expression in the endosperm, molecular assays were performed on the transformed plants. Southern blotting results showed that the GS5 gene was integrated into the maize genome in a low copy number, and RT-PCR analysis showed that the exogenous GS5 gene was normally and highly expressed in maize. The agronomic traits of two successive generations showed that certain lines were significantly improved in yield-related traits, and the most significant changes were observed in the OE-34 line, where the kernel width increased significantly by 8.99% and 10.96%, the 100-kernel weight increased by 14.10% and 10.82%, and the ear weight increased by 13.96% and 15.71%, respectively; however, no significant differences were observed in the plant height, ear height, kernel length, kernel row number, or kernel number. In addition, the overexpression of the GS5 gene increased the grain grouting rate and affected starch synthesis in the rice grains. The kernels’ starch content in OE-25, OE-34, and OE-57 increased by 10.30%, 7.39%, and 6.39%, respectively. Scanning electron microscopy was performed to observe changes in the starch granule size, and the starch granule diameter of the transgenic line(s) was significantly reduced. RT-PCR was performed to detect the expression levels of related genes in starch synthesis, and the expression of these genes was generally upregulated. It was speculated that the exogenous GS5 gene changed the size of the starch granules by regulating the expression of related genes in the starch synthesis pathway, thus increasing the starch content. The trans-GS5 gene was able to be stably expressed in the hybrids with the genetic backgrounds of the four materials, with significant increases in the kernel width, 100-kernel weight, and ear weight. In this study, the maize kernel size was significantly increased through the endosperm-specific expression of the rice GS5 gene, and good material for the functional analysis of the GS5 gene was created, which was of great importance in theory and application. Full article
(This article belongs to the Section Transgenic Technology)
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