ABSTRACT
The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used around the world in both agricultural and non-agricultural fields due to its high activity. However, the heavy use of 2,4-D has resulted in serious environmental contamination, posing a significant risk to non-target organisms, including human beings. This has raised substantial concerns regarding its impact. In addition to agricultural use, accidental spills of 2,4-D can pose serious threats to human health and the ecosystem, emphasizing the importance of prompt pollution remediation. A variety of technologies have been developed to remove 2,4-D residues from the environment, such as incineration, adsorption, ozonation, photodegradation, the photo-Fenton process, and microbial degradation. Compared with traditional physical and chemical remediation methods, microorganisms are the most effective way to remediate 2,4-D pollution because of their rich species, wide distribution, and diverse metabolic pathways. Numerous studies demonstrate that the degradation of 2,4-D in the environment is primarily driven by enzymatic processes carried out by soil microorganisms. To date, a number of bacterial and fungal strains associated with 2,4-D biodegradation have been isolated, such as Sphingomonas, Pseudomonas, Cupriavidus, Achromobacter, Ochrobactrum, Mortierella, and Umbelopsis. Moreover, several key enzymes and genes responsible for 2,4-D biodegradation are also being identified. However, further in-depth research based on multi-omics is needed to elaborate their role in the evolution of novel catabolic pathways and the microbial degradation of 2,4-D. Here, this review provides a comprehensive analysis of recent progress on elucidating the degradation mechanisms of the herbicide 2,4-D, including the microbial strains responsible for its degradation, the enzymes participating in its degradation, and the associated genetic components. Furthermore, it explores the complex biochemical pathways and molecular mechanisms involved in the biodegradation of 2,4-D. In addition, molecular docking techniques are employed to identify crucial amino acids within an alpha-ketoglutarate-dependent 2,4-D dioxygenase that interacts with 2,4-D, thereby offering valuable insights that can inform the development of effective strategies for the biological remediation of this herbicide.
Subject(s)
2,4-Dichlorophenoxyacetic Acid , Biodegradation, Environmental , Herbicides , 2,4-Dichlorophenoxyacetic Acid/metabolism , 2,4-Dichlorophenoxyacetic Acid/chemistry , Herbicides/metabolism , Herbicides/chemistry , Bacteria/metabolism , Bacteria/genetics , Soil Microbiology , Fungi/metabolism , Fungi/geneticsABSTRACT
Bone homeostasis relies on the delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The casein kinase 2 interacting protein-1 (CKIP-1), a specific CK2α subunit-interacting protein, has been documented as one of the crucial negative regulators of bone formation. CKIP-1 siRNA therapy has constraints that limit its use in clinical applications. Therefore, it is necessary to explore effective targeting strategies for CKIP-1. In this study, we observed an upregulation of CKIP-1 protein expression in the microgravity environment, while its ubiquitination levels decreased. We further investigated the interaction between CKIP-1 and VHL and found that VHL enhanced CKIP-1 degradation through the ubiquitylation-proteasome system (UPS). Additionally, we discovered a small molecule ligand, named C77, through DNA-encoded library (DEL) screening, which binds to CKIP-1 both in vivo and in vitro, as confirmed by Surface Plasmon Resonance (SPR) and the Cellular Thermal shift assay (CETSA), respectively. Our findings demonstrated the potential of VHL and C77 as guiding factors in the development of CKIP-1-based Proteolysis-Targeting Chimeras (PROTACs), which could be future therapeutic interventions in disuse osteoporosis.
Subject(s)
Osteoporosis , Von Hippel-Lindau Tumor Suppressor Protein , Humans , Ligands , Osteoporosis/metabolism , Osteoporosis/drug therapy , Osteoporosis/therapy , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Ubiquitination , Carrier Proteins/metabolism , Carrier Proteins/genetics , Proteolysis , Animals , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Mice , Intracellular Signaling Peptides and ProteinsABSTRACT
The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.
ABSTRACT
Pulmonary fibrosis (PF) is one of the sequelae of Corona Virus Disease 2019 (COVID-19), and currently, lung transplantation is the only viable treatment option. Hence, other effective treatments are urgently required. We investigated the therapeutic effects of an approved botanical drug, cepharanthine (CEP), in a cell culture model of transforming growth factor-ß1 (TGF-ß1) and bleomycin (BLM)-induced pulmonary fibrosis rat models both in vitro and in vivo. In this study, CEP and pirfenidone (PFD) suppressed BLM-induced lung tissue inflammation, proliferation of blue collagen fibers, and damage to lung structures in vivo. Furthermore, we also found increased collagen deposition marked by α-smooth muscle actin (α-SMA) and Collagen Type I Alpha 1 (COL1A1), which was significantly alleviated by the addition of PFD and CEP. Moreover, we elucidated the underlying mechanism of CEP against PF in vitro. Various assays confirmed that CEP reduced the viability and migration and promoted apoptosis of myofibroblasts. The expression levels of myofibroblast markers, including COL1A1, vimentin, α-SMA, and Matrix Metallopeptidase 2 (MMP2), were also suppressed by CEP. Simultaneously, CEP significantly suppressed the elevated Phospho-NF-κB p65 (p-p65)/NF-κB p65 (p65) ratio, NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels, and elevated inhibitor of NF-κB Alpha (IκBα) degradation and reversed the progression of PF. Hence, our study demonstrated that CEP prevented myofibroblast activation and treated BLM-induced pulmonary fibrosis in a dose-dependent manner by regulating nuclear factor kappa-B (NF-κB)/ NLRP3 signaling, thereby suggesting that CEP has potential clinical application in pulmonary fibrosis in the future.
Subject(s)
COVID-19 , Pulmonary Fibrosis , Animals , Rats , Bleomycin , Collagen/metabolism , COVID-19/metabolism , Fibroblasts/metabolism , Inflammation/metabolism , Lung , Myofibroblasts/metabolism , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
The genus Triplophysa is an ideal taxon for understanding geological evolution. In this study, for the first time, we report the complete mitochondrial genome of T. nanpanjiangensis Zhu and Cao 1988 using the Nanopore sequencing. It is a circular genome with a length of 16558 bp, comprising 22 tRNAs, 13 protein-coding genes (PCGs), two rRNAs, and one non-coding control region. The phylogenetic tree demonstrates that T. nanpanjiangensis is sister to Triplophysa zhenfengensis and placed within the genus Triplophysa. Our mitogenomic studies provide a new pathway for understanding the molecular phylogeny of the genus Triplophysa.
ABSTRACT
Metagenomic next-generation sequencing (mNGS) is a rapid deep-sequencing diagnostic tool for the unbiased identification of pathogens. In this study, we established a nanopore-sequencing-based mNGS protocol to detect two major viral pathogens of swine, Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV). Samples were spiked with the serially diluted viruses as standard references to define the specific protocols. The utility of the method was evaluated with key parameters. The limits of detection for PRRSV and PEDV were 2.3 × 102 and 9.0 × 104 copies per reaction, respectively, and good correlations between PCR quantification cycle value and the mapped read count (log value) were observed. Only the nanopore reads could be assembled de novo into nearly full-length of the PRRSV genome, with 99.9% pairwise identity, and 90.0% genome coverage for PEDV. The established protocol was validated in PRRSV-positive clinical samples. The results for PRRSV-positive tissue and serum samples tested with mNGS protocol were 100% concordant with quantitative PCR results. The protocol also recognized infections of single or multiple viruses in a single sample. In conclusion, we have established a nanopore-sequencing-based mNGS protocol that efficiently identifies and characterizes viral pathogen(s) in a variety of clinical sample types.
Subject(s)
Nanopore Sequencing , Porcine epidemic diarrhea virus , Porcine respiratory and reproductive syndrome virus , Viruses , Animals , High-Throughput Nucleotide Sequencing/methods , Metagenomics/methods , Porcine epidemic diarrhea virus/genetics , Sensitivity and Specificity , SwineABSTRACT
The E3 ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination processes are closely associated with the occurrence and development of colonic inflammation. Ovarian tumor deubiquitinase 1 (OTUD1) is involved in immunoregulatory functions linked to infectious diseases. However, the effect of OTUD1 on intestinal immune responses during colonic inflammatory disorders such as inflammatory bowel disease (IBD) remains unclear. Here, we show that loss of OTUD1 in mice contributes to the pathogenesis of dextran sulfate sodium (DSS)-induced colitis via excessive release of proinflammatory cytokines. In addition, bone marrow transplantation experiments revealed that OTUD1 in hematopoietic cells plays a dominant role in protection against colitis. Mechanistically, OTUD1 physically interacts with receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and selectively cleaves K63-linked polyubiquitin chains from RIPK1 to inhibit the recruitment of NF-κB essential modulator (NEMO). Moreover, the expression of OTUD1 in mucosa samples from ulcerative colitis (UC) patients was lower than that in mucosa samples from healthy controls. Furthermore, we demonstrate that the UC-associated OTUD1 G430V mutation abolishes the ability of OTUD1 to inhibit RIPK1-mediated NF-κB activation and intestinal inflammation. Taken together, our study unveils a previously unexplored role of OTUD1 in moderating intestinal inflammation by inhibiting RIPK1-mediated NF-κB activation, suggesting that the OTUD1-RIPK1 axis could be a potential target for the treatment of IBD.
Subject(s)
Colitis, Ulcerative , Inflammatory Bowel Diseases , Animals , Colitis, Ulcerative/pathology , Deubiquitinating Enzymes/metabolism , Dextran Sulfate , Humans , Inflammation , Mice , NF-kappa B/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolismABSTRACT
BACKGROUND: Litter size is an important factor that significantly affects the development of the sheep industry. Our previous TMT proteomics analysis found that three key proteins in the ovarian steroidogenesis pathway, STAR, HSD3B1, and CYP11A1, may affect the litter size trait of Small Tail Han sheep. OBJECTIVE: The purpose of this study was to better understand the relationship between polymorphisms of these three genes and litter size. MATERIAL AND METHOD: Sequenom MassARRAY detected genetic variance of the three genes in 768 sheep. Real-time qPCR of the three genes was used to compare their expression in monotocous and polytocous sheep in relevant tissues. Finally, bioinformatics analysis predicted the protein sequences of the different SNP variants. RESULT: Association analysis showed that there was a significant difference in litter size among the genotypes at two loci of the CYP11A1 gene (p < 0.05), but no significant difference was observed in litter size among all genotypes at all loci of the STAR and HSD3B1 genes (p > 0.05). However, STAR expression was significantly different in polytocous and monotocous sheep in the pituitary (p < 0.01). Tissue-specific expression in the ovary was observed for HSD3B1 (p < 0.05), but its expression was not different between polytocous and monotocous sheep. Bioinformatics analysis showed that the g.33217408C > T mutation of CYP11A1 resulted in major changes to the secondary and tertiary structures. In contrast, gene polymorphisms in STAR and HSD3B1 had minimal impacts on their protein structures. DISCUSSION: This may explain why the CYP11A1 variant impacted litter size while the others did not. The single nucleotide polymorphism of the CYP11A1 gene would serve as a good molecular marker when breeding to increase litter size in sheep. Our study provides a basis for further revealing the function of the ovarian steroidogenesis pathway in sheep reproduction and sheep breeding.
Subject(s)
Gene Expression , Ovary/metabolism , Polymorphism, Genetic , Sheep, Domestic/genetics , Steroids/biosynthesis , Animals , Female , Metabolic Networks and Pathways , Polymorphism, Single Nucleotide , Sheep, Domestic/metabolismABSTRACT
Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed.
ABSTRACT
We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.
Subject(s)
Oncogene Proteins, Viral/biosynthesis , Receptor, Notch1/biosynthesis , Repressor Proteins/biosynthesis , Uterine Cervical Neoplasms/drug therapy , Valproic Acid/administration & dosage , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Diamines/administration & dosage , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Viral/drug effects , HeLa Cells/drug effects , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Humans , Oncogene Proteins, Viral/genetics , Receptor, Notch1/genetics , Repressor Proteins/genetics , Snail Family Transcription Factors/biosynthesis , Thiazoles/administration & dosage , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/virologyABSTRACT
The complete mitochondrial genome of the hybrid of Cyprinus carpio L. mirror (â)× Cyprinus Carpio var. singuonensis (â) was characterized first in this study. The total length of the genome was identical to the female parent as 16,581 bp, and the overall base composition was 31.80% A, 24.85% T, 27.55% C and 15.80% G, with a slight A + T bias. It contained 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions (the control region and the origin of the light-strand replication). This study discovered the 99.70% sequence identity between the hybrid and its female parent, which confirmed the maternal inheritance pattern followed by the mitochondrial genome of the hybrid. However, the sequence alignment of mitochondrial genomes between the hybrid and its female parent revealed a total of 47 variable sites in 15 genes or regions, especially 25 sense mutations in 6 PCGs. The complete mitochondrial genome sequence of the hybrid Cyprinus capio Furong may provide an important dataset for further study in mitochondrial inheritance mechanism.
ABSTRACT
The complete mitochondrial genome of the hybrid of Cyprinus capio Furong (â) × Carassius auratus red var. (â) was characterized first in this study. The total length of the mitochondrial genome of Furong-crucian was identical to the female parent as 16,581 bp, and the overall base composition was 31.87% A, 24.81% T, 27.56% C and 15.76% G, with a slight A + T bias. It contained 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions (the control region and the origin of the light-strand replication). This study discovered the 99.3% sequence identity between the hybrid and its female parent, which confirmed the maternal inheritance pattern followed by the mitochondrial genome of the hybrid. However, the sequence alignment of mitochondrial genomes between the hybrid and its female parent revealed a total of 109 variable sites in 18 genes or regions, especially 32 sense mutations in 9 PCGs. The complete mitochondrial genome sequence of this hybrid Furong-crucian may provide an important dataset for further study in mitochondrial inheritance mechanism.
ABSTRACT
OBJECTIVE: To investigate the correlation between extracellular matrix protein-1 (ECM1) and the growth, metastasis and angiogenesis of laryngeal carcinoma. MATERIALS AND METHODS: Forty-five samples with laryngeal benign and malignant tumors confirmed by pathology in Laiwu City People's Hospital from March 2006 to March 2011 were collected, in which there were 29 cases with laryngeal carcinoma and 16 with benign tumors. The expression of ECM1 and factor VIII-related antigens in patients with laryngeal carcinoma and those with benign tumors was respectively detected using immunohistochemical method, and the correlation between ECM1 staining grade and microvessel density (MVD) was analyzed. RESULTS: In laryngeal carcinoma tissue, ECM1 was mainly expressed in cytoplasm, less in cytomembrane or intercellular substance. With abundant expression in the tissue of laryngeal benign tumors (benign mesenchymoma and hemangioma), ECM1 was primarily expressed in the connective tissue, which was different from the expression in laryngeal carcinoma tissue. The proportion of positive ECM1 staining (++) in patients with laryngeal carcinoma was dramatically higher than those with benign tumors (p<0.05), and that of strongly-positive ECM1 staining (+++) slightly higher. The results of Spearman nonparametric correlation analysis revealed that ECM1 staining grade in laryngeal carcinoma tissue had a significantly-positive correlation with MVD (r=0.866, p=0.000). CONCLUSIONS: ECM1 expression in laryngeal carcinoma is closely associated with tumor cell growth, metastasis and angiogenesis, which can be considered as an effective predictor in the occurrence and postoperative recurrence of laryngeal carcinoma.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/pathology , Extracellular Matrix Proteins/metabolism , Laryngeal Neoplasms/pathology , Larynx/metabolism , Microvessels/pathology , Neovascularization, Pathologic/pathology , Adolescent , Adult , Aged , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/metabolism , Case-Control Studies , Child , Female , Follow-Up Studies , Humans , Immunoenzyme Techniques , Laryngeal Neoplasms/blood supply , Laryngeal Neoplasms/metabolism , Lymphatic Metastasis , Male , Microvessels/metabolism , Middle Aged , Neoplasm Invasiveness , Neoplasm Staging , Neovascularization, Pathologic/metabolism , Prognosis , Young AdultABSTRACT
Infection with Giardia is the most commonly diagnosed parasitic cause of diarrhea in the developed world, yet no vaccine exists for human use and a commercially available veterinary vaccine is of limited utility. We have used the adult C57BL/6 mouse model of infection with Giardia duodenalis to better understand immunity to secondary infections with this parasite. Mice were primed by infection with either the GS or WB strains of Giardia and treated with metronidazole on day 7-12 to eliminate the primary infections. Challenge infections on day 21 or day 60 after the primary infections resulted in ~50-fold fewer parasites at day 5 than were found in unprimed mice that only received the challenge infection. Resistance to challenge infections was also observed in B cell deficient µMT mice and when primed mice were challenged with parasites of a different strain. While primed mice developed IgA, mast cell, and T cell responses against the parasite, no specific responses correlated with protection against challenge infections. Together these data suggest that development of an effective vaccine for giardiasis should be feasible since strong immunity can be developed against reinfection in the adult mouse model. Moreover we show that antibody responses are not essential for a protective vaccine and that protection is not parasite strain-specific.
Subject(s)
Disease Resistance , Giardia lamblia/immunology , Giardiasis/immunology , Protozoan Vaccines/immunology , Animals , Antibodies, Protozoan/analysis , Disease Models, Animal , Female , Immunoglobulin A/analysis , Mast Cells/immunology , Mice, Inbred C57BL , Protozoan Vaccines/administration & dosage , Recurrence , T-Lymphocytes/immunologyABSTRACT
Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.
Subject(s)
Foot-and-Mouth Disease Virus/genetics , Foot-and-Mouth Disease/genetics , Foot-and-Mouth Disease/prevention & control , Gene Expression Regulation, Viral , RNA, Small Interfering/genetics , Viral Proteins/genetics , Animals , Animals, Genetically Modified , Animals, Suckling , Cell Line , Conserved Sequence , Disease Resistance , Foot-and-Mouth Disease/virology , Foot-and-Mouth Disease Virus/immunology , Mice , Plasmids/genetics , Plasmids/immunology , Polymerase Chain Reaction , RNA, Small Interfering/metabolism , Swine , Viral Proteins/chemistry , Viral Proteins/immunologyABSTRACT
PURPOSE: We investigated the effects of the anti-epilepsy drug valproic acid (VPA) alone and in combination in treating cervical cancer. METHODS: VPA was investigated for its effects on cervical cancer Hela cell proliferation and tumor growth via in vitro and in vivo assays. RESULTS: VPA induce cell growth suppression and cell cycle arrest, with an increase of Notch1 that acts as a tumor suppressor and the change of other tumor-associated genes such as p21, p63 and PCNA. VPA was also found to induce cell morphological change, with an increase of certain cell transformation markers such as snail1, snail2 and N-cadherin. Moreover, VPA could significantly up-regulate somatostatin receptor type II (SSTR2). Our in vivo study further demonstrated that VPA via inducing SSTR2 up-regulation extremely enhanced the anti-tumor ability of the SSTR2-preferential cytotoxic COL-SST conjugate in xenografts. CONCLUSIONS: VPA could not only suppress tumor progression but also provide a novel promising therapeutic choice in combination with a receptor-targeted cytotoxic conjugate via activating the specific receptor.
Subject(s)
Anticonvulsants/pharmacology , Carcinoma/drug therapy , Receptor, Notch1/metabolism , Receptors, Somatostatin/metabolism , Signal Transduction/drug effects , Valproic Acid/pharmacology , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Colchicine/therapeutic use , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclooxygenase 2/metabolism , Drug Combinations , G2 Phase Cell Cycle Checkpoints/drug effects , Gene Expression , Genes, p53/drug effects , HeLa Cells , Hormones/therapeutic use , Humans , Membrane Proteins/genetics , Mice , Mice, Nude , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , RNA, Messenger/metabolism , Receptor, Notch1/genetics , Somatostatin/therapeutic use , Tubulin Modulators/therapeutic useABSTRACT
BACKGROUND: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS: We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA. RESULTS: Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-ß and TNF-α in transfected cells. CONCLUSIONS: Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.
Subject(s)
HSC70 Heat-Shock Proteins/metabolism , Hepatitis B virus/genetics , Hepatitis B virus/physiology , RNA Interference , RNA, Small Interfering/genetics , Viral Proteins/metabolism , Cell Line , Flow Cytometry , Gene Expression Regulation, Viral , Gene Silencing , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSC70 Heat-Shock Proteins/genetics , Hep G2 Cells , Hepatitis B/virology , Hepatitis B virus/metabolism , Humans , Plasmids , Transfection , Viral Proteins/genetics , Virus Replication/geneticsABSTRACT
Interferon-δ (IFN-δ) belongs to type I IFN. It was first identified by Lefevre in 1993. The porcine interferon-δ (PoIFN-δ)-related sequences were reported in 2009. The IFN-δ-related sequences have not been studied as closely as IFN-α and IFN-ß. In this study, we identified 11 functional genes and 7 pseudogenes of the PoIFN-δ-related subtypes from the porcine genome database. Three of these genes were cloned, expressed, and functionally studied. PoIFN-δ-related subtypes can bind to the type I IFN receptors, activate the Janus kinase-signal transduce and activator of transcription (JAK-STAT) signaling pathway, induce IFN stimulate genes, and other cytokines. The antiviral activity of PoIFN-δ-related subtypes varies a lot in different subtypes. Among all the PoIFN-δ subtypes detected, PoIFN-δ8 has the strongest antiviral and immunomodulatory activities.
Subject(s)
Genome , Interferon Regulatory Factors/genetics , Interferon Type I/metabolism , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Cloning, Molecular , Cricetinae , Cytokines/genetics , Gene Expression , Genetic Loci , Interferon Regulatory Factors/metabolism , Interferon Type I/chemistry , Interferon Type I/genetics , Janus Kinases/metabolism , Molecular Docking Simulation , Phylogeny , Protein Binding , Receptor, Interferon alpha-beta/chemistry , Receptor, Interferon alpha-beta/metabolism , STAT Transcription Factors/metabolism , Signal Transduction , Swine/genetics , Swine/metabolismABSTRACT
OBJECTIVE: To explore the antiviral efficacy of small interfering RNAs (siRNAs)/shRNA targeting preC/C of HBV in human hepatoma cells Huh-7 and HepG2.2.15 cells. METHODS: Three 21 nucleotide(nt) siRNAs for treating HBV preC/C gene were designed and synthesized according to the HBV genome in GenBank accession numbers (U95551); simultaneously, one 21-nt-long non-homologous siRNA was also designed randomly for negative control. They were cloned into vector pU6 for constructing shRNA-expressing plasmids pU6-C1, pU6-C2, pU6-C3 and control pU6-C4. To assess the function of siRNAs, a reporter gene system was constructed. The HBV preC/C gene was synthesized by PCR with pT-HBV1.3 as the template. The preC/C gene was then inserted into the enhanced green fluorescent protein expression vector (EGFP-N1) in order to construct the recombinant plasmid pEGFP-preC/C (E-C), which carries the EGFP reporter gene. The three shRNA-expressing plasmids-pU6-C1, pU6-C2, or pU6-C3-was each then cotransfected into Huh-7 cells along with either reporter gene expression vector E-C or the controls; or these three plasmids-pU6-C1, pU6-C2, or pU6-C3-was each cotransfected into HepG2.2.15 cells along with the controls. First, upon determination of the number of cells exhibiting EGFP expression in Huh-7cells as detected by an BH-2 fluorescence microscope and FACS-440 flow cytometry at different times after cotransfection, the investigators evaluated the inhibitory efficiency of the three shRNA-expressing plasmids by an EGFP reporter system in cultured cells. Subsequently, the expression amount of HBsAg and HBeAg in HepG2.2.15 cell supernatant at 24, 48, 72 and 96 h post-cotransfection was detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the expression of HBsAg and HBcAg at 72 h post-cotransfection in HepG2.2.15 cells. The copy level of HBV mRNA transcripts cDNA in HepG2.2.15 cells was further investigated through quantitative real-time polymerase chain reaction (real-time PCR). RESULTS: In comparison with single plasmid transfection pEGFP-N1 or E-C, fluorescence microscope examination and flow cytometry detection at 48 hours after cotransfection indicated that the expression of the reporter gene EGFP in cotransfected group Huh-7 cell involving pU6-C1, pU6-C2 or pU6-C3 resulted in an 80% reduction in EGFP signal relative to the controls (P < 0.01). It was also found through immunofluorescence that the expression of HBsAg and HBcAg in HepG2.2.15 cells was reduced markedly (P < 0.01), that the copy level of HBV mRNA transcripts cDNA as detected at 48 hours after cotransfection by quantitative real-time PCR was reduced respectively by 73.9% ± 1.2% (P = 0.029), 48.2% ± 1.8% and 35.8% ± 1.4% (P = 0.037, 0.040) relative to the control, that it conformed with that detected by fluorescence microscope/flow cytometry, ELISA, and immunofluorescence (P < 0.01). Thereby further corroborating the antiviral efficacy of RNAi. The efficacy was obvious at 48 h, reaching a peak at 72 h. CONCLUSION: For the first time it has been found that RNAi induced by siRNA/shRNA targeting HBV preC/C gene is effective and specific in inhibiting HBV replication and expression in human hepatoma cells Huh-7 and HepG2.2.15 cells. Our data suggest that RNAi may provide an effective, viable approach in gene therapy to treating major infectious diseases such as HBV/HCV/HIV infection.
Subject(s)
Carcinoma, Hepatocellular/virology , Hepatitis B virus/physiology , Liver Neoplasms/virology , RNA Interference , Gene Expression Regulation, Viral , Gene Targeting , Genetic Vectors , Hep G2 Cells , Hepatitis B virus/genetics , Humans , RNA, Small Interfering/genetics , Virus ReplicationABSTRACT
OBJECTIVE: The vaccines currently developed against infectious diseases fail to induce effective antiviral immune responses to control an abrupt outbreak of viral diseases epidemic in a short space of time. Hence there is an urgent need to develop emergency vaccines capable of producing prophylactic effects against infectious diseases. RNA interference (RNAi) is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes. The rapidity and uniqueness of RNAi responses can make up for the current inadequacy of antiviral preventive regimes. Here we evaluate the antiviral potential of short hairpin RNA (shRNA) targeting C (core) gene of hepatitis B virus (HBV). It plays essential roles in HBcAg encoding and viral attachment to susceptible cells during its life cycle. The present study was intended to investigate the inhibitory effect of C-specific shRNAs on HBV replication and expression in BHK-21 cells. METHODS: The reporter gene expression vector of pC-EGFP-N1 was constructed by cloning the DNA (PCR product) of HBV C into the EcoRI-HindIII sites of pEGFP-N1 to fuse C to enhanced green fluorescent protein (EGFP) for providing a reporting system for monitoring siRNA function. Plasmid pC was constructed by cloning the DNA of HBV C into the EcoRI-HindIII sites of pCDNA3.1B(-) directly under the control of cytomegalovirus promoter. Two plasmids (S1 & S2) were constructed to express shRNAs targeting C of HBV with the length of 24 nucleotide (nt) homologous in sequence to the HBV C gene. Plasmids were designed and synthesized according to the HBV genome (HBV genotype B, ayw1 subtype) of chronic hepatic B patients from 56 ethnic minorities in China. After cloning and sequencing, the investigators registered them with the GenBank accession numbers of AY517488 (CYN/2002) and AY517489 (CYN/2000), etc. Simultaneously, one of nonspecific shRNA-S3 with a length of 24 nt was also designed randomly for negative control. After cloning into vector pU6 for constructing shRNA-expressing plasmids, they were then cotransfected into BHK-21 cells along with reporter gene expression vector of pC-EGFP-N1. First, upon a determination of the number of cells exhibiting EGFP expression in BHK-21 cells as detected by an Olympus BH-2 fluorescence microscope and FACS-440 flow cytometry (Becton-Dickinson, USA) at different times after cotransfection, the investigators evaluated the gene inhibitory efficiency of both plasmids by an EGFP reporter system in BHK-21 cells. Subsequently, the antiviral efficacy of both plasmids in BHK-21 cells was further investigated by real-time quantitative polymerase chain reaction. RESULTS: In comparison with single plasmid transfection pC-EGFP-N1 or pEGFP-N1, fluorescence microscope and flow cytometry detection at 24 h post-cotransfection revealed that the expression of reporter gene EGFP in cotransfection group involving S1 or S2 and S1 + S2 cotransfection group was reduced significantly by about 90% in EGFP signal versus the control. And the EGFP expression in control plasmid cotransfected S3 or pU6 was not significantly reduced in BHK-21 cells (P < 0.01). It was found that the expression of mRNAs of HBV C and EGFP gene as detected by real-time quantitative PCR was the same as that detected by fluorescence microscope and flow cytometry (P < 0.01), thereby further corroborating the antiviral efficacy of RNAi. The antiviral efficacy extended to almost 48 hours. The results indicted that a DNA vector-based RNAi technology could effectively and specifically inhibit the replication and expression of HBV in BHK-21 cells. CONCLUSION: For the first time it has been found that RNAi induced by shRNA targeting C gene exerts an effective and unique inhibition of HBV replication and expression in BHK-21 cells. Thus RNAi may provide an effective emergency vaccine against major infectious diseases such as HBV infection.