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Keywords = full-length molecular clone

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18 pages, 4945 KiB  
Article
Overexpression of a White Clover WRKY Transcription Factor Improves Cold Tolerance in Arabidopsis
by Shuaixian Li, Meiyan Guo, Wei Hong, Manman Li, Xiaoyue Zhu, Changhong Guo and Yongjun Shu
Agronomy 2025, 15(7), 1700; https://doi.org/10.3390/agronomy15071700 - 14 Jul 2025
Viewed by 349
Abstract
Plants are frequently exposed to various abiotic stresses, among which low-temperature stress markedly impairs growth and physiological functions. WRKY transcription factors are key regulators in plant responses to abiotic stress. In this study, a novel WRKY transcription factor gene, TrWRKY79, was cloned [...] Read more.
Plants are frequently exposed to various abiotic stresses, among which low-temperature stress markedly impairs growth and physiological functions. WRKY transcription factors are key regulators in plant responses to abiotic stress. In this study, a novel WRKY transcription factor gene, TrWRKY79, was cloned from white clover. Functional characterization revealed that the full-length TrWRKY79 protein possesses typical features of transcription factors, including transcriptional activation activity located at its C-terminal domain. Heterologous expression of TrWRKY79 in Arabidopsis thaliana significantly enhanced cold tolerance under low-temperature stress. Physiological assays showed that the transgenic lines exhibited higher chlorophyll content and elevated activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) compared to wild-type plants. Furthermore, Protenix was employed to predict the potential target genes of TrWRKY transcription factors, and their expression profiles were analyzed to help elucidate the regulatory network underlying cold tolerance. qRT-PCR analysis confirmed that several cold-responsive genes, such as COR47 and ABI5, were significantly upregulated in the transgenic lines. Collectively, these findings indicate that TrWRKY79 plays a positive regulatory role in enhancing cold tolerance, providing valuable insights into the molecular mechanisms of cold resistance in white clover and offering promising candidate genes for improving stress resilience in forage crops. Full article
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13 pages, 14235 KiB  
Article
Expression and Biological Activity Analysis of Recombinant Fibronectin3 Protein in Bacillus subtilis
by Chaozheng Lu, Guangxin Xu, Yin Tian, Zhiwei Yi and Xixiang Tang
BioTech 2025, 14(3), 51; https://doi.org/10.3390/biotech14030051 - 23 Jun 2025
Viewed by 406
Abstract
Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction [...] Read more.
Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction with integrin receptors on the cell surface. However, the large molecular weight of wild-type FN presents challenges for its large-scale production through heterologous expression. Therefore, this study focused on cloning the FN3 functional domain of full-length FN for expression and validation. This study selected Bacillus subtilis as the expression host due to its prominent advantages, including efficient protein secretion, absence of endotoxins, and minimal codon bias. The recombinant vector pHT43-FN3 was successfully constructed through homologous recombination technology and transformed into Bacillus subtilis WB800N. The FN3 protein was successfully expressed after induction with IPTG. Following purification of the recombinant FN protein using a His-tag nickel column, SDS-PAGE analysis showed that the molecular weight of FN3 was approximately 27.3 kDa. Western blot analysis confirmed the correct expression of FN3, and the BCA protein assay kit determined a protein yield of 5.4 mg/L. CCK8 testing demonstrated the good biocompatibility of FN3. In vitro cell experiments showed that FN3 significantly promoted cell migration at a 20 μg/mL concentration and enhanced cell adhesion at 10 μg/mL. In summary, this study successfully utilized Bacillus subtilis to express the FN3 functional domain peptide from FN protein and has validated its ability to promote cell migration and adhesion. These findings not only provide a strategy for the expression of FN protein in B. subtilis, but also establish an experimental foundation for the potential application of FN3 protein in tissue repair fields such as cutaneous wound healing and cartilage regeneration. Full article
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12 pages, 2331 KiB  
Article
Regulation of Petal Coloration by the Auxin Amide Hydrolase Gene RhILL1 in Rose (Rosa hybrida)
by Dan Wang, Yiping Zhang, Daliang Li, Xujun Ma, Xiao Yang, Hongying Jian, Huichun Wang, Lihua Wang, Hao Zhang, Qigang Wang and Xianqin Qiu
Genes 2025, 16(6), 691; https://doi.org/10.3390/genes16060691 - 6 Jun 2025
Viewed by 566
Abstract
Objective: This study aimed to elucidate the regulatory mechanism of an auxin amide hydrolase gene (IAA-Leucine Resistant1-like Hydrolase, RhILL1) in the petal pigmentation of rose (Rosa hybrida), providing theoretical insight into the hormonal regulation of flower coloration at the molecular [...] Read more.
Objective: This study aimed to elucidate the regulatory mechanism of an auxin amide hydrolase gene (IAA-Leucine Resistant1-like Hydrolase, RhILL1) in the petal pigmentation of rose (Rosa hybrida), providing theoretical insight into the hormonal regulation of flower coloration at the molecular level. Methods: Using petals at Stage 3 (S3) of the cut rose cultivar ‘Pink Floyd’ as experimental material, we cloned the rose auxin amide hydrolase gene RhILL1 and validated its function via virus-induced gene silencing (VIGS). The expression levels of anthocyanin biosynthetic genes, anthocyanin content, and auxin (IAA) levels were analyzed to assess the role of RhILL1 in petal pigmentation. Results: The full-length open reading frame (ORF) of RhILL1 was cloned, spanning 1326 bp and encoding a 441-amino-acid protein harboring two conserved domains, Peptidase_M20 and M20_dimer, characteristic of the ILL1 protein family. Functional characterization was performed using VIGS. Quantitative real-time PCR (qRT-PCR) revealed that RhILL1 expression progressively increased from the Green (G) stage to S3, correlating with intensified petal coloration. Silencing RhILL1 resulted in visibly lighter petals, the reduced expression of anthocyanin biosynthetic genes, and a significant decrease in endogenous indole-3-acetic acid (IAA) levels compared with controls. Moreover, exogenous application of 10 μM naphthaleneacetic acid (NAA) to petals significantly preserved petal pigmentation. Conclusion: These findings suggest that RhILL1 contributes to the development and maintenance of petal coloration in rose, likely by modulating IAA levels, thereby influencing the expression of anthocyanin biosynthesis-related genes. Full article
(This article belongs to the Section Plant Genetics and Genomics)
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15 pages, 4256 KiB  
Article
Cloning and Spatiotemporal Expression Analysis of IGF1R Gene cDNA in Alopex lagopus (Arctic Fox)
by Wei Xu, Hualin Fu, Xiangyu Meng, Yiwen Sun, Fangyong Ning and Zhiheng Du
Life 2025, 15(5), 796; https://doi.org/10.3390/life15050796 - 17 May 2025
Viewed by 417
Abstract
This study aimed to clarify the sequence characteristics and spatiotemporal expression patterns of the insulin-like growth factor 1 receptor (IGF1R) gene in Alopex lagopus (Arctic fox), thereby addressing the existing knowledge gap regarding IGF1R-mediated growth regulation in this species. The [...] Read more.
This study aimed to clarify the sequence characteristics and spatiotemporal expression patterns of the insulin-like growth factor 1 receptor (IGF1R) gene in Alopex lagopus (Arctic fox), thereby addressing the existing knowledge gap regarding IGF1R-mediated growth regulation in this species. The findings establish a crucial foundation for subsequent investigations into the correlation between this gene and Arctic fox growth traits. Specific primers were designed based on the cDNA sequence of the canine IGF1R gene (Accession No. XM_545828). The full-length coding sequence (CDS) of the Arctic fox IGF1R gene (1617 bp, encoding 538 amino acids) was successfully cloned via RT-PCR. Phylogenetic analysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm revealed a 99% sequence homology in the IGF1R gene between the Arctic fox and canine, confirmed their closest evolutionary relationship. Protein characterization showed that the IGF1R protein has a molecular weight of 60.62 kDa (theoretical isoelectric point pI = 5.15), containing one fibronectin type-III domain and one tyrosine kinase domain, classifying it as an acidic hydrophilic transmembrane protein. Phosphorylation site prediction identified 27 phosphorylation sites, with secondary structures dominated by α-helices (26.39%) and random coils (52.79%). The IGF1R gene displayed significant tissue-specific expression variations across 12 examined tissues in Arctic foxes: highest expression levels in testis, minimal expression in stomach, and no detectable expression in duodenum. Spatiotemporal expression analysis revealed that in 2-, 4-, and 6-month-old individuals, hepatic IGF1R exhibited a progressive increase, testicular expression reached peak levels at 6 months, and skeletal muscle demonstrated transient upregulation peaking at 4 months. These spatiotemporal expression patterns suggest that IGF1R may participate in metabolism and organ developmental processes during critical growth stages of Arctic foxes through tissue-specific regulation. Full article
(This article belongs to the Section Animal Science)
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15 pages, 3977 KiB  
Article
Characterization and Functional Analysis of Trim38 in the Immune Response of the Large Yellow Croaker (Larimichthys crocea) Against Pseudomonas plecoglossicida Infection
by Qiaoying Li, Hongling Wu, Ying Huang, Dinaer Yekefenhazi, Wenzheng Zou and Fang Han
Int. J. Mol. Sci. 2025, 26(9), 4150; https://doi.org/10.3390/ijms26094150 - 27 Apr 2025
Viewed by 471
Abstract
The large yellow croaker (Larimichthys crocea) is a cornerstone species in Chinese marine aquaculture, yet bacterial infections—particularly visceral white nodules disease (VWND) caused by Pseudomonas plecoglossicida—severely compromise its production. This study aimed to elucidate the immunoregulatory mechanisms of tripartite motif-containing [...] Read more.
The large yellow croaker (Larimichthys crocea) is a cornerstone species in Chinese marine aquaculture, yet bacterial infections—particularly visceral white nodules disease (VWND) caused by Pseudomonas plecoglossicida—severely compromise its production. This study aimed to elucidate the immunoregulatory mechanisms of tripartite motif-containing protein 38 in the large yellow croaker (Lctrim38) during bacterial infections, with an emphasis on host–pathogen interactions involving P. plecoglossicida, to evaluate its potential for disease-resistant breeding applications. The full-length cDNA of Lctrim38 was cloned and characterized, with structural analysis revealing a conserved domain architecture comprising RING, B-box, coiled-coil, and PRY-SPRY motifs. Functional characterization through Lctrim38 overexpression in large yellow croaker kidney cells (PCK cells) demonstrated significant modulation of key immune-related pathways, including TGF-β, PI3K-Akt, IL-17, and PPAR. Notably, Lctrim38-mediated inhibition of NF-κB signaling was shown to downregulate pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ), establishing its role as a negative regulator of inflammatory responses. These findings provide insights into the immune mechanisms of Trim38 in large yellow croakers and highlight its potential as a molecular target for disease resistance breeding. Future research should explore its broader functions, including its antiviral potential. Full article
(This article belongs to the Section Molecular Immunology)
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13 pages, 4655 KiB  
Article
Cloning, Expression, and Bioinformatics Analysis of the AvFD1 Gene in Amomum villosum Lour
by Duo Wang, Yating Zhu, Shuang Li, Hongyou Zhao, Chongnan Wang, Qianxia Li, Yanfang Wang, Chunyong Yang, Ge Li, Yanqian Wang and Lixia Zhang
Biology 2025, 14(5), 457; https://doi.org/10.3390/biology14050457 - 24 Apr 2025
Viewed by 445
Abstract
As a perennial medicinal plant in the Zingiber genus, Amomum villosum Lour. faces agricultural challenges due to its prolonged vegetative and reproductive growth phases, which hinder efficient pollination and delay fruiting. To address this limitation, the present study aimed to identify the FD [...] Read more.
As a perennial medicinal plant in the Zingiber genus, Amomum villosum Lour. faces agricultural challenges due to its prolonged vegetative and reproductive growth phases, which hinder efficient pollination and delay fruiting. To address this limitation, the present study aimed to identify the FD gene involved in regulating flowering in A. villosum to provide a basis for research on the molecular mechanisms of early fruiting cultivars. Based on the differentially expressed gene AvFD1 obtained from the transcriptome database of early fruiting plants and controls, specific primers were designed for PCR to clone the full-length sequence of AvFD1. The characteristics of the cloned AvFD1 gene were analyzed using online bioinformatics software. The expression profiles of AvFD1 in various tissues and in 1- and 2-year bearing A. villosum varieties were investigated by quantitative real-time PCR. This study successfully cloned the FD1 gene sequence of A. villosum, marking the first reported characterization of this gene in the species. Tissue-specific expression analysis revealed significantly elevated AvFD1 expression levels in stolon tips and flower buds compared to tender leaves, suggesting its potential role as a positive regulator of flowering initiation. The obtained sequence establishes essential molecular data for subsequent functional validation of AvFD1 in A. villosum. Full article
(This article belongs to the Special Issue Young Investigators in Biochemistry and Molecular Biology)
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14 pages, 8990 KiB  
Article
Molecular Cloning and Heterologous Expression of the Mitochondrial ATP6 Gene from Kenaf (Hibiscus cannabinus) in Tobacco (Nicotiana tabacum)
by Bangbang Huang, Meiling Wei, Rongchang Wei, Wenhuan Hou, Xingfu Tang, Yanhong Zhao, Xiaofang Liao and Ruiyang Zhou
Genes 2025, 16(5), 479; https://doi.org/10.3390/genes16050479 - 23 Apr 2025
Viewed by 483
Abstract
Background: The aim of this study was to develop a genetic transformation system to construct an overexpression vector for the mitochondrial gene atp6 in tobacco, thereby providing a foundation to investigate the functional roles of mitochondrial genes in this species. Methods: A full-length [...] Read more.
Background: The aim of this study was to develop a genetic transformation system to construct an overexpression vector for the mitochondrial gene atp6 in tobacco, thereby providing a foundation to investigate the functional roles of mitochondrial genes in this species. Methods: A full-length coding sequence (CDS) of the atp6 gene from a sterile line was cloned, along with the mitochondrial leader peptide sequence of atp2-1 from tobacco, using cDNA from kenaf UG93A anthers as a template. An overexpression vector for plants was constructed by employing In-Fusion technology, and wild-type tobacco plants were transformed via Agrobacterium-mediated transformation. Transgenic tobacco plants were then subjected to resistance screening and PCR validation. Results: The overexpression vector PBI121-atp2-1-atp6-EGFP, which includeds the mitochondrial leader peptide sequence, was successfully constructed. PCR validation using two pairs of primers targeting different sites on the overexpression vector confirmed the stable expression of the target gene in six transgenic tobacco plants (H1, H3, H4, H5, H7, and H8) via both primer pairs. A phenotypic analysis and iodine–potassium iodide (I2-KI) staining of pollen grains from transgenic tobacco plants revealed the presence of shriveled and malformed pollen grains with reduced viability. These findings suggest that the atp6A gene, including the mitochondrial signal peptide, induces pollen abortion in tobacco. Conclusions: The genetic transformation system developed for the vector overexpressing the atp6 mitochondrial gene from kenaf provides a valuable framework to investigate the molecular regulatory mechanisms underlying the role of the atp6 gene in kenaf cytoplasmic male sterility (CMS). Full article
(This article belongs to the Section Plant Genetics and Genomics)
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18 pages, 8564 KiB  
Article
The Role of AaPGRP-LB in the Immune Response of Aedes albopictus Against Bacteria Infection
by Cheng Wu, Yahui Chen, Chenhua Zheng, Xitong Huang, Yuyang Xie, Lingqun Lin, Xiuli Zhang and Lihua Xie
Int. J. Mol. Sci. 2025, 26(5), 2188; https://doi.org/10.3390/ijms26052188 - 28 Feb 2025
Viewed by 703
Abstract
The initial phase of an insect’s innate immune response to foreign pathogens is triggered by the identification of exogenous invaders, a mechanism facilitated by pattern recognition receptors. Among these receptors, peptidoglycan recognition proteins (PGRPs), abundant in insects, are essential components of the innate [...] Read more.
The initial phase of an insect’s innate immune response to foreign pathogens is triggered by the identification of exogenous invaders, a mechanism facilitated by pattern recognition receptors. Among these receptors, peptidoglycan recognition proteins (PGRPs), abundant in insects, are essential components of the innate immune system. The roles of PGRPs have been extensively elucidated in Drosophila melanogaster; however, the mechanism underlying the immune response of Aedes albopictus to pathogens is unclear. Herein, we successfully cloned the full-length cDNA of a PGRP gene from Ae. albopictus, designated as the AaPGRP-LB gene. The open reading frame of AaPGRP-LB encodes 203 amino acids, including a secretion signal peptide and a canonical PGRP conserved domain. Multisequence alignment revealed that AaPGRP-LB possesses the amino acid residues essential for zinc binding and amidase activity. Molecular docking studies demonstrated that AaPGRP-LB exhibits a strong binding affinity for DAP-type and LYS-type peptidoglycan. The mRNA expression level of the AaPGRP-LB gene significantly increased after oral infection with Escherichia coli or Staphylococcus aureus. The purified recombinant AaPGRP-LB (rAaPGRP-LB) exhibited strong agglutination properties and demonstrated significant antimicrobial efficacy against E. coli and S. aureus in the presence of zinc ions. This study highlights the critical role of AaPGRP-LB in the immune response of Ae. albopictus. These findings provide a foundation for future research on mosquito immune pathways for innovative vector control and disease prevention strategies. Full article
(This article belongs to the Section Molecular Immunology)
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14 pages, 3665 KiB  
Article
Molecular Characterization of BsCu/Zn-Superoxide Dismutases and BsMn-Superoxide Dismutases from Chinese Black Sleeper (Bostrychus sinensis)
by Haolin Li, Suzhen Ran, Xiaoyu Wang, Bin Shen and Jianshe Zhang
Animals 2025, 15(5), 653; https://doi.org/10.3390/ani15050653 - 24 Feb 2025
Viewed by 454
Abstract
Superoxide dismutases (SODs) are critical antioxidant enzymes that protect organisms by catalyzing the dismutation of superoxide radicals into molecular oxygen (O2) and hydrogen peroxide (H2O2). In this study, we cloned the full-length cDNA of two SODs (BsCu/Zn-SOD [...] Read more.
Superoxide dismutases (SODs) are critical antioxidant enzymes that protect organisms by catalyzing the dismutation of superoxide radicals into molecular oxygen (O2) and hydrogen peroxide (H2O2). In this study, we cloned the full-length cDNA of two SODs (BsCu/Zn-SOD and BsMn-SOD) from the Chinese black sleeper. The cDNA of BsCu/Zn-SOD consists of 465 nucleotides encoding 154 amino acids, while the BsMn-SOD comprises 678 nucleotides encoding 225 amino acids. The PCR results indicated that in normal tissues, the highest expression of BsCu/Zn-SOD (1.02-fold) was primarily observed in muscle, whereas the highest expression of BsMn-SOD (6.42-fold) was noted in skin. Following treatment with Vibrio parahaemolyticus, the highest expression of BsCu/Zn-SOD (3.67-fold) was detected in the spleen, while BsMn-SOD (3.36-fold) presented the highest expression in the blood. After poly(I:C) treatment, the highest expression of BsCu/Zn-SOD (2.92-fold) was found in the blood, with BsMn-SOD (2.37-fold) showing the highest expression in the spleen. Following Cd2+ treatment, the highest expression of BsCu/Zn-SOD (3.36-fold) was recorded in the liver at the 1/2 96 h LC50 concentration, and in the spleen at both the 1/4 96 h LC50 (2.50-fold) and 1/8 96 h LC50 concentrations. In contrast, BsMn-SOD presented the highest expression (2.60-fold) in the liver across all three concentrations. These findings suggest that both BsCu/Zn-SOD and BsMn-SOD play vital roles in the innate immune response to environmental challenges, laying the foundation for further exploration of antioxidant mechanisms. Full article
(This article belongs to the Section Aquatic Animals)
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12 pages, 2311 KiB  
Article
Genomic Characterization of Laodelphax striatellus Permutotetra-like Virus and Self-Cleavage Function of Viral Capsid Protein
by Jun Piao, Jiarui Zhang, Lujie Zhang, Jingai Piao, Haitao Wang, Yilin Xie and Shuo Li
Microbiol. Res. 2025, 16(1), 9; https://doi.org/10.3390/microbiolres16010009 - 2 Jan 2025
Viewed by 890
Abstract
Laodelphax striatellus permutotetra-like virus (LsPLV) is a novel insect virus identified via small RNA deep sequencing. At present, there is a lack of awareness of LsPLV, restricting research on its utilization in biocontrol. In this paper, the full-length genome of LsPLV was cloned [...] Read more.
Laodelphax striatellus permutotetra-like virus (LsPLV) is a novel insect virus identified via small RNA deep sequencing. At present, there is a lack of awareness of LsPLV, restricting research on its utilization in biocontrol. In this paper, the full-length genome of LsPLV was cloned and analyzed, then viral capsid protein (CP) was expressed and prepared as an antibody, and CP property was tested. It was found that the LsPLV genome was 4667 nt in length, encoding two proteins, RNA-dependent RNA polymerase (RdRP) and CP, and the palm subdomain conserved region in RdRp was arranged in a “C–A–B” permutation pattern, exhibiting the typical characteristics of permutotetra-like viruses. Phylogenetic analysis suggested that LsPLV shared the highest homology (excluding LsPLV1) with a Nodaviridae virus (QLI47702.1), and their nucleotide identities of RdRP and CP were 55.4% and 59.2%, respectively. After expression, purified CP exhibited two bands of 60 kDa and 47 kDa, suggesting a potential cleavage in the protein. LsPLV CP in L. striatellus was detected by Western blot, and except for the complete CP band, the specific bands with molecular weights lower than CP were also detected, indicating that CP underwent cleavage. Detection of purified CP in vitro showed that the cleavage could occur independent of any protease, confirming that CP has self-cleavage characteristics. Full article
(This article belongs to the Special Issue Veterinary Microbiology and Diagnostics)
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18 pages, 2215 KiB  
Article
Virulence and Replicative Fitness of HIV-1 Transmitted/Founder (T/F) Viruses Harbouring Drug Resistance-Associated Mutation
by Aanand Sonawane, Deepak Selvam, Ling Yue, Manohar Nesakumar, Sandhya Vivekanandan, Manickam Ashokkumar, Eric Hunter and Luke Elizabeth Hanna
Viruses 2024, 16(12), 1854; https://doi.org/10.3390/v16121854 - 29 Nov 2024
Viewed by 1242
Abstract
The biological characteristics of early transmitted/founder (T/F) variants are crucial factors for viral transmission and constitute key determinants for the development of better therapeutics and vaccine strategies. The present study aimed to generate T/F viruses and to characterize their biological properties. For this [...] Read more.
The biological characteristics of early transmitted/founder (T/F) variants are crucial factors for viral transmission and constitute key determinants for the development of better therapeutics and vaccine strategies. The present study aimed to generate T/F viruses and to characterize their biological properties. For this purpose, we constructed 18 full-length infectious molecular clones (IMCs) of HIV from recently infected infants. All the clones were characterized genotypically through whole genome sequencing and phenotypically for infectivity, replication kinetics, co-receptor usage, as well as their susceptibility to neutralizing antibodies and entry inhibitors using standard virological assays. Genotypic analysis revealed that all the T/F clones were of non-recombinant subtype C, but some of them harboured the Y181C drug resistance mutation associated with resistance to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretroviral drugs. In vitro studies showed that while all the IMCs were capable of replicating in PBMCs and utilized the CCR5 co-receptor for cellular entry, the drug-resistant variants had significantly lower replicative capacity and per particle infectivity than the drug-sensitive viruses. Both exhibited similar sensitivities to a standard panel of broadly neutralizing monoclonal antibodies and viral entry inhibitors. These findings suggest that despite their diminished replicative fitness, the drug-resistant T/F variants retain transmission fitness and remain susceptible to neutralizing antibody-based interventions and viral entry inhibitors. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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22 pages, 7524 KiB  
Article
The Molecular Mechanism of Farnesoid X Receptor Alleviating Glucose Intolerance in Turbot (Scophthalmus maximus)
by Gaochan Qin, Mingzhu Pan, Dong Huang, Xinxin Li, Yue Liu, Xiaojun Yu, Kangsen Mai and Wenbing Zhang
Cells 2024, 13(23), 1949; https://doi.org/10.3390/cells13231949 - 23 Nov 2024
Cited by 1 | Viewed by 1008
Abstract
To explore the molecular targets for regulating glucose metabolism in carnivorous fish, the turbot (Scophthalmus maximus) was selected as the research object to study. Farnesoid X receptor (FXR; NR1H4), as a ligand-activated transcription factor, plays an important role in glucose metabolism [...] Read more.
To explore the molecular targets for regulating glucose metabolism in carnivorous fish, the turbot (Scophthalmus maximus) was selected as the research object to study. Farnesoid X receptor (FXR; NR1H4), as a ligand-activated transcription factor, plays an important role in glucose metabolism in mammals. However, the mechanisms controlling glucose metabolism mediated by FXR in fish are not understood. It was first found that the protein levels of FXR and its target gene, small heterodimer partner (SHP), were significantly decreased in the high-glucose group (50 mM, HG) compared with those in the normal glucose group (15 mM, CON) in primary hepatocytes of turbot. By further exploring the function of FXR in turbot, the full length of FXR in turbot was cloned, and its nuclear localization function was characterized by subcellular localization. The results revealed that the FXR had the highest expression in the liver, and its capability to activate SHP expression through heterodimer formation with retinoid X receptor (RXR) was proved, which proved RXR could bind to 15 binding sites of FXR by forming hydrogen bonds. Activation of FXR in both the CON and HG groups significantly increased the expression of glucokinase (gk) and pyruvate kinase (pk), while it decreased the expression of cytosolic phosphoenolpyruvate carboxykinase (cpepck), mitochondrial phosphoenolpyruvate carboxykinase (mpepck), glucose-6-phosphatase 1 (g6pase1) and glucose-6-phosphatase 2 (g6pase2), and caused no significant different in glycogen synthetase (gs). ELISA experiments further demonstrated that under the condition of high glucose with activated FXR, it could significantly decrease the activity of PEPCK and G6PASE in hepatocytes. In a dual-luciferase reporter assay, the FXR could significantly inhibit the activity of G6PASE2 and cPEPCK promoters by binding to the binding site ‘ATGACCT’. Knockdown of SHP after activation of FXR reduced the inhibitory effect on gluconeogenesis. In summary, FXR can bind to the mpepck and g6pase2 promoters to inhibit their expression, thereby directly inhibiting the gluconeogenesis pathway. FXR can also indirectly inhibit the gluconeogenesis pathway by activating shp. These findings suggest the possibility of FXR as a molecular target to regulate glucose homeostasis in turbot. Full article
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16 pages, 2701 KiB  
Article
The Molecular Characterization and Antioxidant Defense of a Novel Nrf2 from the Pacific Abalone Haliotis discus hannai Ino
by Kun Qiao, Qiongmei Huang, Bei Chen, Min Xu, Hua Hao, Yongchang Su, Shuji Liu, Nan Pan and Zhiyu Liu
Int. J. Mol. Sci. 2024, 25(22), 12429; https://doi.org/10.3390/ijms252212429 - 19 Nov 2024
Cited by 1 | Viewed by 1013
Abstract
The Nrf2/ARE pathway is considered the most important endogenous antioxidant signaling pathway in mammals, playing a crucial role in defending against external damage. This study investigated the functional characteristics of Nrf2 in the abalone, Haliotis discus hannai. The full-length cDNA sequence of [...] Read more.
The Nrf2/ARE pathway is considered the most important endogenous antioxidant signaling pathway in mammals, playing a crucial role in defending against external damage. This study investigated the functional characteristics of Nrf2 in the abalone, Haliotis discus hannai. The full-length cDNA sequence of the HdhNrf2 gene was cloned using rapid amplification of cDNA ends (RACE) technology and consists of 4568 base pairs encoding a protein of 694 amino acids. The predicted theoretical molecular weight was 77 kDa, with an isoelectric point of 4.72. Multiple sequence alignment analysis revealed the relative conservation of the HdhNrf2 amino acid sequence in H. discus hannai. The tissue expression pattern of the HdhNrf2 gene was analyzed using real-time fluorescence quantitative PCR, which showed the highest expression in the gills, followed by hemocytes, with the lowest levels in the foot and mantle. The inducible expression of HdhNrf2 and antioxidant genes in abalone under H2O2 stress was investigated at various time points. Furthermore, an expression vector, pET-28a(+)-rHdhNrf2, was constructed, and the recombinant protein rHdhNrf2 was obtained through induced expression and purification. These findings indicated that HdhNrf2 plays a crucial role in the defense of abalones against oxidative stress. Full article
(This article belongs to the Section Molecular Biology)
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14 pages, 2130 KiB  
Article
Comparative Characterization of Three Homologous Glutathione Transferases from the Weed Lolium perenne
by Annie Kontouri, Farid Shokry Ataya, Panagiotis Madesis and Nikolaos Labrou
Foods 2024, 13(22), 3584; https://doi.org/10.3390/foods13223584 - 9 Nov 2024
Viewed by 1124
Abstract
The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes [...] Read more.
The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide. Full article
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15 pages, 9809 KiB  
Article
Cloning and Identification of Common Carp (Cyprinus carpio) PI3KC3 and Its Expression in Response to CyHV-3 Infection
by Xiaona Jiang, Lijing Tian, Wanying Ren, Chitao Li, Xuesong Hu, Yanlong Ge, Lei Cheng, Xiaodan Shi and Zhiying Jia
Curr. Issues Mol. Biol. 2024, 46(10), 11714-11728; https://doi.org/10.3390/cimb46100696 - 21 Oct 2024
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Abstract
Phosphoinositide 3-kinases (PI3Ks) are a class of key regulatory factors in eukaryotes that can inhibit viral replication by influencing autophagy. Currently, cyprinid herpesvirus 3 (CyHV-3) poses a serious threat to common carp culture. However, PI3K has not yet been identified in common carp. [...] Read more.
Phosphoinositide 3-kinases (PI3Ks) are a class of key regulatory factors in eukaryotes that can inhibit viral replication by influencing autophagy. Currently, cyprinid herpesvirus 3 (CyHV-3) poses a serious threat to common carp culture. However, PI3K has not yet been identified in common carp. In this study, full-length PI3KC3 from common carp (CcPI3KC3), consisting of an open reading frame (ORF) of 2664 bp encoding a polypeptide of 887 amino acids, with a predicted molecular mass of 101.19 kDa and a theoretical isoelectric point (pI) of 5.97, was cloned. The amino acid and nucleotide sequences of CcPI3KC3 displayed high similarity to yellow catfish’s (Tachysurus fulvidraco) PI3KC3. The tissue expression profile revealed that the mRNA levels of CcPI3KC3 in the liver, spleen, and head kidney were significantly greater than those in the brain, heart, intestines, gills, eyes, testes, and ovaries of common carp. We compared the expression patterns of CcPI3KC3 between “Longke-11” mirror carp (CyHV-3-resistant carp) and German mirror carp (non-resistant to CyHV-3) at different times (0, 48, 96, 144 h, 192, 240, 288 h post-infection (hpi)) after CyHV-3 infection. The results revealed that CcPI3KC3 mRNA expression significantly increased in the early infection stage. In the CyHV-3-resistant mirror carp variety, the relative expression of CcPI3KC3 was significantly greater at 48, 96, and 144 hpi compared with the nonbreeding strain groups after infection (p < 0.001). These results indicate that the full-length CcPI3KC3 sequence was successfully cloned from common carp for the first time, and it might play an important role in the immune system of common carp against CyHV-3 infection. This study provides a theoretical basis for the molecular mechanism of CyHV-3 resistance. Full article
(This article belongs to the Special Issue Research on Virus-Induced Cellular and Molecular Responses)
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