Targeting Smurf1 to block PDK1-Akt signaling in KRAS-mutated colorectal cancer.

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.

The Discovery of a Specific CKIP-1 Ligand for the Potential Treatment of Disuse Osteoporosis.

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.

Microorganism-Driven 2,4-D Biodegradation: Current Status and Emerging Opportunities.

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.

Cepharanthine Ameliorates Pulmonary Fibrosis by Inhibiting the NF-κB/NLRP3 Pathway, Fibroblast-to-Myofibroblast Transition and Inflammation.

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.

Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice.

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.

Valproic acid suppresses cervical cancer tumor progression possibly via activating Notch1 signaling and enhances receptor-targeted cancer chemotherapeutic via activating somatostatin receptor type II.

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.

The complete mitochondrial genome of Triplophysa nanpanjiangensis Zhu and Cao 1988 (Cypriniformes: Nemacheilidae).

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.

Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70.

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.

[PreC/C gene-targeting RNA interference suppresses hepatitis B virus replication and expression in human hepatoma cells].

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.

Rapid metagenomic identification of two major swine pathogens with real-time nanopore sequencing.

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.

The deubiquitinase OTUD1 inhibits colonic inflammation by suppressing RIPK1-mediated NF-κB signaling.

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.

Polymorphism, expression and structure analysis of key genes in the ovarian steroidogenesis pathway in sheep (Ovis aries).

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.

[Expressions of bFGF and PTEN in cervical carcinoma and their clinical significance].

OBJECTIVE: To investigate the expressions of bFGF and PTEN in cervical carcinoma and their clinical significance. METHODS: Tissue microarray technique and immunohistochemistry SP method were used to detect the expressions of bFGF and PTEN in 143 cases of invasive carcinoma of cervix (ICC) and 20 cases of normal cervical epithelium remote from tumor (NCE). The relationship between the expressions of bFGF and PTEN in ICC and some factors relating to clinical pathology of cervical carcinoma such as histopathological grading, lymph node metastasis, stroma involvement and FIGO staging were analyzed. RESULTS: The rate of the positive expression of bFGF in ICC was significantly higher than that in NCE 88.8% (127/143) vs. 25.0% (5/20, P = 0.000). The rate of positive expression of PTEN in ICC was significantly lower than that in NCE 67.1% (96/143) vs. 100.0% (20/20, P = 0.000). The expression of bFGF was positively correlated with lymph node metastasis and histopathological grading (r = 0.239, P = 0.004 and r = 0.369, P = 0.000, respectively). The expression of PTEN was negatively correlated with FIGO staging, histopathological grading and lymph node metastasis (r = -0.189, P = 0.024; r = -0.211, P = 0.011; r = -0.321, P = 0.000, respectively). The expression of bFGF was negatively correlated with the expression of PTEN in ICC (r = -0.261, P = 0.002). CONCLUSION: The overexpression of bFGF and underexpression of PTEN are closely related to the invasion and growth of cervical carcinoma. Detection of the expression of both bFGF and PTEN may be of value in further understanding the biological behavior and predicting the prognosis of cervical carcinoma.

[C gene-targeting short hairpin RNA suppresses the replication and expression of hepatitis B virus in BHK-21 cells].

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.

Proteomic analysis of serum in lung cancer induced by 3-methylcholanthrene.

Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. To determine the differently expressed proteins in the serum of lung cancer and figure out the function of the proteins, two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to screen the serum proteins of lung cancer model induced by 3-methylcholanthrene (MCA). From optimized 2DE image, 455 spots in the normal sera and 716 spots in the lung cancers sera were detected. Among them, 141 protein spots were differentially expressed when comparing the serum from normal rat and serum from lung cancer model, including 82 overexpressed proteins and 59 underexpressed proteins. Changes of haptoglobin, transthyretin, and TNF superfamily member 8 (TNFRS8) were confirmed in sera from lung cancer by MALDI-TOF-MS. Proteomics technology leads to identify changes of haptoglobin, transthyretin, and TNFRS8 in serum of rat lung cancer model and represents a powerful tool in searching for candidate proteins as biomarkers.

[Transfection of siRNA expressing plasmids targeting S gene inhibits replication and expression of hepatitis B virus in hepatic cancer cells].

OBJECTIVE: To study the inhibitive effects of transfection of siRNA expressing plasmids targeting S gene, one of the 4 open reading frames of hepatitis B virus (HBV), on the replication and expression of HBV. METHODS: Two plasmids expressing 2 siRNAs targeting S gene, one of the 4 open reading frames of HBV (S1 and S2) and one nonspecific plasmid (siRNA-S3), as negative control, with the length of 21 nt heterologous to the HBV/U95551 genome were constructed, and then transfected into the hepatic cancer cells of the line HepG2.2.15. 48 hours later, real-time PCR was used to evaluate the gene silencing efficiency and ELISA was used to detect the expression of HBsAg and hepatitis B e antigen (HBeAg), protein markers of HBV, in the supernatants. RESULTS: The inhibition rates of HBsAg and HBeAg expression of the HepG2.2.15 cells transfected with S1 were 60% and 56% respectively, those of the HepG2.2.15 cells transfected with S2 were 73% and 70% respectively, those of the HepG2.2.15 cells transfected with S1+S2 were 82% and 78% respectively, and those of the HepG2.2.15 cells transfected with S3 were not significantly different from those of the blank control group. RT-PCR showed that the mRNA expression rates of HBsAg and HBeAg in the HepG2.2.15 cells transfected with S1, S2, and S1+S2 were inhibited by 64%-88% t respectively. CONCLUSION: Transfection of the vector plasmids expressing the siRNAs targeting S gene inhibits the expression of HBsAg and HBeAg in hepatic cancer cells. RNAi may provide a viable strategy against viruses for the prevention and treatment of HBV infection.

Detection of differential expression of porcine IFN-alpha subtypes by reverse transcription polymerase chain reaction.

The porcine interferon-alpha (IFN-alpha) multigene family is a new IFN-alpha system in recent research. Characterization of the PoIFN-alpha multigene family has been described in our previous work, and 14 functional PoIFN-alpha genes were detected in the porcine genome. In this study, we designed subtype-specific primers and consensus primers for PoIFN-alpha. The expression of PoIFN-alpha was detected using the two PCR strategies in three systems, namely, poly(I).poly(C)-DEAE-dextran-induced PK15 cells, pseudorabies virus-infected PK15 cells, and infected PK15 cells with an attenuated strain of swine fever virus, respectively. In poly(I).poly(C)-DEAE-dextran-induced PK15 cells, the expression of IFN-alpha2, -alpha3, -alpha4, -alpha8, and -alpha9 after 6-h/24-h inducement in PK15 cells were observed. In pseudorabies virus-infected PK15 cells, the expression of PoIFN-alpha2, -alpha3, -alpha8, -alpha9, -alpha10, and -alpha13 was observed after 6-h/24-h infection, and in the attenuated strain of swine fever virus-infected PK15 cells, upregulation of PoIFN-alpha2, -alpha3, -alpha4, -alpha8, -alpha9, and -alpha10 was detected. The results of realtime quantitative PCR analysis suggested that the expression was time-dependent in pseudorabies virus/poly(I).poly(C)-DEAE-dextran-induced PK15 cells, but in the attenuated swine fever virus-infected PK15 system, the expression level of IFN-alpha subtypes was not obviously time dependent.

Induction of lung lesions in Wistar rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its inhibition by aspirin and phenethyl isothiocyanate.

BACKGROUND: The development of effective chemopreventive agents against cigarette smoke-induced lung cancer could be greatly facilitated by suitable laboratory animal models, such as animals treated with the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the current study, we established a novel lung cancer model in Wistar rats treated with NNK. Using this model, we assessed the effects of two chemopreventive agents, aspirin and phenethyl isothiocyanate (PEITC), on tumor progression. METHODS: First, rats were treated with a single-dose of NNK by intratracheal instillation; control rats received iodized oil. The animals were then sacrificed on the indicated day after drug administration and examined for tumors in the target organs. PCNA, p63 and COX-2 expression were analyzed in the preneoplastic lung lesions. Second, rats were treated with a single-dose of NNK (25 mg/kg body weight) in the absence or presence of aspirin and/or PEITC in the daily diet. The control group received only the vehicle in the regular diet. The animals were sacrificed on day 91 after bronchial instillation of NNK. Lungs were collected and processed for histopathological and immunohistochemical assays. RESULTS: NNK induced preneoplastic lesions in lungs, including 33.3% alveolar hyperplasia and 55.6% alveolar atypical dysplasia. COX-2 expression increased similarly in alveolar hyperplasia and alveolar atypical dysplasia, while PCNA expression increased more significantly in the latter than the former. No p63 expression was detected in the preneoplastic lesions. In the second study, the incidences of alveolar atypical dysplasia were reduced to 10%, 10% and 0%, respectively, in the aspirin, PEITC and aspirin and PEITC groups, compared with 62.5% in the carcinogen-treated control group. COX-2 expression decreased after dietary aspirin or aspirin and PEITC treatment. PCNA expression was significantly reduced in the aspirin and PEITC group. CONCLUSION: (1) A single dose of 25 mg/kg body weight NNK by intratracheal instillation is sufficient to induce preneoplastic lesions in Wistar rat lungs. (2) COX-2 takes part in NNK-induced tumorigenesis but is not involved in proliferation. (3) Aspirin and PEITC have protective effects in the early stages of tumor progression initiated by NNK.

A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide.

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.

Characterization of the porcine alpha interferon multigene family.

The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-alpha (PoIFN-alpha) multigene family. Fourteen functional PoIFN-alpha genes and two PoIFN-alpha pseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes. A phylogenetic tree of the porcine IFN-alpha gene family defined the evolutionary relationship of the various subtypes. In addition, analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome. Eight PoIFN-alpha subtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line. We detected the level of transcription by real-time quantitative RT-PCR analysis. The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus (VSV) and PK 15 cells/Pseudorabies Virus (PRV) respectively. We found the antiviral activities of intact PoIFN-alpha genes are approximately 2-50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15-55 times higher in PK 15 cells. There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility.