Revelation of rice molecular design breeding: the blend of tradition and modernity.

As one of the major staple crops, rice feeds more than one half of the world population. Due to increasing population and dramatic climate change, the rice varieties with higher yield performance and excellent overall agronomic performance should be developed. The raise of molecular design breeding concept provides opportunity to get new breakthrough for variety development, and it is important to clarify the efficient gene combination during actual breeding. In this review, we summarize the recent advances about rice variety improvement either by marker assisted selection (MAS) breeding or popular gene editing technique, which will be beneficial to understand different aspects of the molecular design breeding. We provide genetic views for the classical MAS application, including the genetic effect of key genes and their combinations, the recurrent genome recovery rate at different backcross generations, linkage drag and recombination selection. Moreover, we compare the breeding value of recently-developed molecular techniques, including the advantage of high-throughput genotyping and the way and effect of gene editing in creating useful traits. Considering the current status and actual demands of rice breeding, we raise the strategy to take advantages of both traditional breeding resources and popular molecular techniques, which might pave the way to optimize the process of molecular design breeding in future.

Retracted: Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis.

This publication has been retracted by the Editor due to the identification of non-original figure images that raise concerns regarding the credibility and originality of the study. Reference: You-Dong Wan, Rui-Xue Zhu, Zhong-Zheng Bian, Xin-Ting Pan. Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitisy. Med Sci Monit, 2019; 25: 4609-4616. DOI: 10.12659/MSM.914538.

Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis.

BACKGROUND Gut microbiota dysbiosis plays a key role in pathogenesis of severe acute pancreatitis (SAP). In this study, we explored the protective effects of the p38 MAPK inhibitor, SB203580, against gut inflammation and microbiota dysbiosis induced by pancreatic duct injection with 3.5% sodium taurocholate in an SAP rat model. MATERIAL AND METHODS Ninety male Sprague-Dawley rats were randomly assigned to sham-operated, SAP model, and SAP plus SB203580 groups (n=30/group). Histological examination was conducted to assess gut and pancreatitis injury. The levels of amylase, D-lactate, diamine oxidase, tumor necrosis factor alpha, IL-6, IL-1ß, and phospho-p38MAPK in the plasma and intestine were evaluated at 3, 6, or 12 h after SAP induction. The gut microbiome was investigated based on16S rDNA gene sequencing at 12 h after SAP induction. RESULTS Histological examination revealed edema and inflammatory infiltrations in the pancreas and distal ileum. The expression of tumor necrosis factor alpha, IL-1ß, and IL-6 in plasma and distal ileum was increased in the SAP group, which were restored after treatment with SB203580. Significantly lower bacterial diversity and richness was found in the SAP group. In the SAP group, the abundance of Bacteroidetes and Firmicutes was decreased, and there was a higher proportion of Proteobacteria at the phylum level. The SAP plus SB203580 group exhibited significantly less damage to the gut microbiota, with higher bacterial diversity and a more normal proportion of intestinal microbiota. CONCLUSIONS SB203580 mediated suppression of the p38 MAPK signaling pathway via reduced gut inflammatory response and microbiota dysbiosis.

Microarray and network-based identification of functional modules and pathways of active tuberculosis.

Diagnose of active tuberculosis (TB) is challenging and treatment response is also difficult to efficiently monitor. The aim of this study was to use an integrated analysis of microarray and network-based method to the samples from publically available datasets to obtain a diagnostic module set and pathways in active TB. Towards this goal, background protein-protein interactions (PPI) network was generated based on global PPI information and gene expression data, following by identification of differential expression network (DEN) from the background PPI network. Then, ego genes were extracted according to the degree features in DEN. Next, module collection was conducted by ego gene expansion based on EgoNet algorithm. After that, differential expression of modules between active TB and controls was evaluated using random permutation test. Finally, biological significance of differential modules was detected by pathways enrichment analysis based on Reactome database, and Fisher's exact test was implemented to extract differential pathways for active TB. Totally, 47 ego genes and 47 candidate modules were identified from the DEN. By setting the cutoff-criteria of gene size >5 and classification accuracy ≥0.9, 7 ego modules (Module 4, Module 7, Module 9, Module 19, Module 25, Module 38 and Module 43) were extracted, and all of them had the statistical significance between active TB and controls. Then, Fisher's exact test was conducted to capture differential pathways for active TB. Interestingly, genes in Module 4, Module 25, Module 38, and Module 43 were enriched in the same pathway, formation of a pool of free 40S subunits. Significant pathway for Module 7 and Module 9 was eukaryotic translation termination, and for Module 19 was nonsense mediated decay enhanced by the exon junction complex (EJC). Accordingly, differential modules and pathways might be potential biomarkers for treating active TB, and provide valuable clues for better understanding of molecular mechanism of active TB.

Inhibition of STAT Pathway Impairs Anti-Hepatitis C Virus Effect of Interferon Alpha.

BACKGROUND/AIMS: Signal transducer and activator of transcription (STAT) pathway plays an important role in antiviral efficacy of interferon alpha (IFN-α). IFN-α is the main therapeutic against hepatitis C virus (HCV) infection. We explored effects of IFN-α on HCV replication and antiviral gene expression by targeting STAT. METHODS: In response to IFN-α, STAT status, HCV replication, and antiviral gene expression were analyzed in human hepatoma Huh7.5.1 cells before and after cell culture-derived HCV infection. RESULTS: IFN-α treatment induced expression and phosphorylation of STAT1 and STAT2 in Huh7.5.1 cells. Pretreatment of Huh7.5.1 cells with a mAb to IFN alpha receptor (IFNAR) 2 decreased IFN-α-dependent phosphorylation of STAT1 and STAT2, whereas pretreatment with an IFNAR1 mAb increased such phosphorylation, suggesting that IFNAR mediates IFN-α-triggered STAT signaling. During HCV infection, STAT1 and STAT2 phosphorylation could be rescued by IFN-α and IFN-α-induced phosphorylation of STAT1 and STAT2 was impaired. Inhibition of STAT pathway by Jak inhibitor I significantly enhanced HCV RNA replication and viral protein expression. Antiviral genes coding for IFN regulatory factor 9 and IFN-stimulated gene 15 were up-regulated by IFN-α during HCV infection but such up-regulation was abrogated by Jak inhibitor I. CONCLUSION: These results establish that activation of STAT pathway is essential for anti-HCV efficacy of IFN-α. Impairment of IFN-α-triggered STAT signaling by HCV may account for evading IFN-α response.

Effects of light quality on the accumulation of phytochemicals in vegetables produced in controlled environments: a review.

Phytochemicals in vegetables are important for human health, and their biosynthesis, metabolism and accumulation are affected by environmental factors. Light condition (light quality, light intensity and photoperiod) is one of the most important environmental variables in regulating vegetable growth, development and phytochemical accumulation, particularly for vegetables produced in controlled environments. With the development of light-emitting diode (LED) technology, the regulation of light environments has become increasingly feasible for the provision of ideal light quality, intensity and photoperiod for protected facilities. In this review, the effects of light quality regulation on phytochemical accumulation in vegetables produced in controlled environments are identified, highlighting the research progress and advantages of LED technology as a light environment regulation tool for modifying phytochemical accumulation in vegetables.

Two ABCI family transporters, OsABCI15 and OsABCI16, are involved in grain-filling in rice.

Seed development is critical for plant reproduction and crop yield, with panicle seed-setting rate, grain-filling, and grain weight being key seed characteristics for yield improvement. However, few genes are known to regulate grain filling. Here, we identify two adenosine triphosphate (ATP)-binding cassette (ABC)I-type transporter genes, OsABCI15 and OsABCI16, involved in rice grain-filling. Both genes are highly expressed in developing seeds, and their proteins are localized to the plasma membrane and cytosol. Interestingly, knockout of OsABCI15 and OsABCI16 results in a significant reduction in seed-setting rate, caused predominantly by the severe empty pericarp phenotype, which differs from the previously reported low seed-setting phenotype resulting from failed pollination. Further analysis indicates that OsABCI15 and OsABCI16 participate in ion homeostasis and likely export ions between filial tissues and maternal tissues during grain filling. Importantly, overexpression of OsABCI15 and OsABCI16 enhances seed-setting rate and grain yield in transgenic plants and decreases ion accumulation in brown rice. Moreover, the OsABCI15/16 orthologues in maize exhibit a similar role in kernel development, as demonstrated by their disruption in transgenic maize. Therefore, our findings reveal the important roles of two ABC transporters in cereal grain filling, highlighting their value in crop yield improvement.

Isojacareubin from the Chinese herb Hypericum japonicum: potent antibacterial and synergistic effects on clinical methicillin-resistant Staphylococcus aureus (MRSA).

Through bioassay-guided fractionation of the extracts from the aerial parts of the Chinese herb Hypericum japonicum Thunb. Murray, Isojacareubin (ISJ) was characterized as a potent antibacterial compound against the clinical methicillin-resistant Staphylococcus aureus (MRSA). The broth microdilution assay was used to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ISJ alone. The results showed that its MICs/MBCs ranged from 4/16 to 16/64 µg/mL, with the concentrations required to inhibit or kill 50% of the strains (MIC(50)/MBC(50)) at 8/16 µg/mL. Synergistic evaluations of this compound with four conventional antibacterial agents representing different types were performed by the chequerboard and time-kill tests. The chequerboard method showed significant synergy effects when ISJ was combined with Ceftazidime (CAZ), Levofloxacin (LEV) and Ampicillin (AMP), with the values of 50% of the fractional inhibitory concentration indices (FICI(50)) at 0.25, 0.37 and 0.37, respectively. Combined bactericidal activities were also observed in the time-kill dynamic assay. The results showed the ability of ISJ to reduce MRSA viable counts by log(10)CFU/mL at 24 h of incubation at a concentration of 1 × MIC were 1.5 (LEV, additivity), 0.92 (CAZ, indifference) and 0.82 (AMP, indifference), respectively. These in vitro anti-MRSA activities of ISJ alone and its synergy with conventional antibacterial agents demonstrated that ISJ enhanced their efficacy, which is of potential use for single and combinatory therapy of patients infected with MRSA.

Antibacterial and synergy of berberines with antibacterial agents against clinical multi-drug resistant isolates of methicillin-resistant Staphylococcus aureus (MRSA).

Antibacterial activity of berberine (Ber) and 8-acetonyl-dihydroberberine (A-Ber) alone and combined uses with antibacterial agents ampicillin (AMP), azithromycin (AZM), cefazolin (CFZ) and levofloxacin (LEV) was studied on 10 clinical isolates of SCCmec III type methicillin-resistant Staphylococcus aureus (MRSA). Susceptibility to each agent alone was tested using a broth microdilution method and the chequerboard and time-kill tests for the combined evaluations, respectively. The alone MICs/MBCs (µg/mL) ranges were 32-128/64-256 (Ber) and 32-128/128-512 (A-Ber). Significant synergies were observed for the Ber (A-Ber)/AZM and Ber (A-Ber)/LEV combinations against 90% of the tested MRSA strains, with fractional inhibitory concentration indices (FICIs) values ranged from 0.188 to 0.500. An additivity result was also observed for the Ber/AZM combination by time-kill curves. These results demonstrated for the first time that Ber and A-Ber enhanced the in vitro inhibitory efficacy of AZM and LEV to a same extent, which had potential for further investigation in combinatory therapeutic applications of patients infected with MRSA.

Evaluation of traditional Chinese medicinal plants for anti-MRSA activity with reference to the treatment record of infectious diseases.

The in vitro antimicrobial activities of 30 Chinese medicinal plants were evaluated with reference to the treatment record of infectious diseases in the Traditional Chinese Medicine (TCM) literature. The plant materials were extracted with 80% ethanol and the extracts were primarily screened against conventional clinical pathogens like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans by the agar diffusion method. Their inhibition zone diameters (IZDs, mm, 50 mg/mL) ranged from 2,048 by the standard broth microdilution method. The seven extracts from M. yunnanensis, S. sinensis, G. morella, E. daneillii, M. squamulata, S. arborescens and B. hancei were determined as the most active extracts, with MICs of 8-64 µg/mL. The results were in good agreement with their traditional applications in skin and other infections.

[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.

Investigation of the Mechanism of 60Co Gamma-Ray Irradiation-Stimulated Oxidation Enhancing the Antigenicity of Ovalbumin by High-Resolution Mass Spectrometry.

60Co gamma-ray irradiation-induced antigenicity changes in ovalbumin (OVA) were investigated, and the molecular mechanism was analyzed. Irradiation treatment at 0-100 kGy could significantly enhance the IgG/IgE binding ability of OVA in a dose-dependent paradigm by concomitant oxidative modification, which exhibited color browning and an increase in carbonyl content caused by high-penetrable rays. More allergenic epitopes of OVA were exposed after irradiation treatment reflected by structural changes including the unfolding of tertiary structure, the conversion of α-helix structures to ß-sheet and random coil structures, and the cleavage of several peptide bonds. Meanwhile, three oxidation sites of K46, T49, and N260 located in key linear epitopes were observed, which might increase the allergenic ability of OVA via the disaggregation of noncovalent bonds and the unwinding of α-helix structures. Conclusively, irradiation may enhance the potential allergenicity of OVA by oxidative modification, which provides theoretical guidance for effectively controlling the oxidation of proteins in the irradiation process.

Effect of probiotics on length of hospitalization in mild acute pancreatitis: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Acute pancreatitis is the leading cause of hospitalization for acute gastrointestinal disease worldwide. The effects of probiotics in mild acute pancreatitis have not been studied. We hypothesized that the administration of probiotics may accelerate the recovery of intestinal function and shorten the length of hospital stay (LOS) in patients with mild pancreatitis. AIM: To investigate the value of probiotics in reducing the LOS in patients with mild acute pancreatitis. METHODS: We conducted a double-blind randomized clinical trial to evaluate the effects of probiotics administered to patients with mild acute pancreatitis at a tertiary medical center. The patients were given probiotics capsules (a mixed preparation of Bacillus subtilis and Enterococcus faecium) or placebo. The primary study endpoint was the LOS. The secondary endpoints included time to abdominal pain relief, recurrent abdominal pain, and time to successful oral feeding. RESULTS: A total of 128 patients were included, with 64 patients in each arm. The severity of illness and the etiological distribution of disease were similar in the two groups. There was a significant reduction in the LOS in the probiotics treatment group vs the placebo group (5.36 ± 0.15 vs 6.02 ± 0.17 d, P < 0.05). The probiotics group was associated with a shorter time to abdominal pain relief and time to successful oral feeding (P < 0.01 for both) than the placebo group. No statistical difference was found in recurrent abdominal pain between the two groups. CONCLUSION: The study results showed that the administration of probiotics capsules is associated with a shorter duration of hospitalization in patients with mild acute pancreatitis.

[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.

Grain Size Selection Using Novel Functional Markers Targeting 14 Genes in Rice.

BACKGROUND: Grain size is an extremely important aspect of rice breeding, affecting both grain yield and quality traits. It is controlled by multiple genes and tracking these genes in breeding schemes should expedite selection of lines with superior grain yield and quality, thus it is essential to develop robust, efficient markers. RESULT: In this study, 14 genes related to grain size (GW2, GS2, qLGY3, GS3, GL3.1, TGW3, GS5, GW5, GS6, TGW6, GW6a, GLW7, GL7 and GW8) were selected for functional marker development. Twenty-one PCR-gel-based markers were developed to genotype the candidate functional nucleotide polymorphisms (FNPs) of these genes, and all markers can effectively recognize the corresponding allele types. To test the allele effects of different FNPs, a global collection of rice cultivars including 257 accessions from the Rice Diversity Panel 1 was used for allele mining, and four grain-size-related traits were investigated at two planting locations. Three FNPs for GW2, GS2 and GL3.1 were genotyped as rare alleles only found in cultivars with notably large grains, and the allele contributions of the remaining FNPs were clarified in both the indica and japonica subspecies. Significant trait contributions were found for most of the FNPs, especially GS3, GW5 and GL7. Of note, GW5 could function as a key regulator to coordinate the performance of other grain size genes. The allele effects of several FNPs were also tested by QTL analysis using an F2 population, and GW5 was further identified as the major locus with the largest contribution to grain width and length to width ratio. CONCLUSIONS: The functional markers are robust for genotyping different cultivars and may facilitate the rational design of grain size to achieve a balance between grain yield and quality in future rice breeding efforts.

Hepatitis C virus F protein inhibits cell apoptosis by activation of intracellular NF-kappaB pathway.

AIM: To observe the influence of HCV F protein on apoptosis of HepG2 cells, and explore the association between F protein and NF-kappaB signal pathway. METHODS: HCV 1b F gene containing HepG2-F cells and HCV 1b C gene containing HepG2-C cells were treated with 100 IU/mL TNF-alpha, and analyzed by flow cytometry, Western blotting, and dual luciferase reporter assay. Empty plasmid pcDNA3.1(+) containing HepG2-3.1 cells were used as control. RESULTS: (i) With the treatment of TNF-alpha for 18 h, the apoptosis rates (AR) of HepG2-F and HepG2-3.1 cells were 0.41% (+/- 0.11%) and 37.43% (+/- 2.03%) respectively, while that of HepG2-C was 4.07% (+/- 0.18%). At 36 h after TNF-alpha treatment, the AR of HepG2-F and HepG2-3.1 cells were 10.03% (+/- 0.41%) and 44.63% (+/- 3.37%), and that of HepG2-C was 14.95% (+/- 0.85%). (ii) After the treatment of TNF-alpha for 0.5-18 h, the p65 contents in the whole cells of HepG2-F and HepG2-3.1 showed no significant difference (P = 0.34, t = 1.08), while the p65 contents in the nucleus of HepG2-F and HepG2-3.1 cells were 3.8-1.9 times and 1.8-1.0 times higher than that in the non-treated cells (P = 0.013, t = 4.25). (iii) The relative luciferase unit (RLU) of the HepG2 cells, co-transfected with pcDNA3.1-F and pNF-kappaB-luc, and then treated with TNF-alpha (100 IU/mL) for 18 h, showed a pcDNA3.1-F dose-dependent increase. CONCLUSION: HCV F protein can over-activate NF-kappaB signal pathway, which makes HepG2-F cells able to resist TNF-alpha induced apoptosis.

[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.

Effects of Ligustrazine on Airway Inflammation in A Mouse Model of Neutrophilic Asthma.

OBJECTIVE: To investigate the effects of ligustrazine (LTZ) on airway inflammation in a mouse model of neutrophilic asthma (NA). METHODS: Forty healthy C57BL/6 female mice were randomly divided into 4 groups using a random number table, including the normal control, NA, LTZ and dexamethasone (DXM) groups, with 10 rats in each group. The NA mice model was established by the method of ovalbumin combined with lipopolysaccharide sensitization. At 0.5 h before each challenge, LTZ and DXM groups were intraperitoneally injected with LTZ (80 mg/kg) or DXM (0.5 mg/kg) for 14 d, respectively, while the other two groups were given the equal volume of normal saline. After last challenge for 24 h, the aerosol inhalation of methacholine was performed and the airway reactivity was measured. The bronchoalveolar lavage fluid (BALF) was collected. The Wright-Giemsa staining was used for total white blood cells and differential counts. The levels of cytokines interleukin (IL)-17 and IL-10 were detected by enzyme-linked immunosorbent assay. The pathological change of lung tissue was observed by hematoxylin eosin staining. RESULTS: The airway responsiveness of the NA group was signifificantly higher than the normal control group (P<0.05), while those in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05). The neutrophil and eosinophil counts in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05), and those in the LTZ group were signifificantly lower than the DXM group (P<0.05). There were a large number of peribronchiolar and perivascular inflammatory cells in fifiltration in the NA group. The airway inflflammation in the LTZ and DXM groups were signifificantly alleviated than the NA group. The infifiltration in the LTZ group was signifificantly reduced than the DXM group. Compared with the normal control group, the IL-17 level in BALF was signifificantly increased and the IL-10 level in BALF was signifificantly decreased in the NA group (P<0.05). LTZ and DXM treatment signifificantly decreased IL-17 levels and increased IL-10 levels compared with the NA group (P<0.05), and the changes in the above indices were more signifificant in the LTZ group (P<0.05). CONCLUSION: LTZ could alleviate the airway inflflammation in the NA mice model through increasing the IL-10 level and decreasing the IL-17 level.

[Expression in Escherichia coli of hepatitis B virus genes from minority nationality patients in Yunnan province with chronic hepatitis B and their antigenicity].

OBJECTIVE: To investigate the expression in Escherichia coli (E. coli) of hepatitis B virus (HBV) genes from minority nationality patients in Yunnan province with chronic hepatitis B (CHB) and their antigenicity. METHODS: Peripheral blood samples were collected from 25 minority nationality patients with CHB in Yunnan province. Twenty-five CHB patients of Han nationality in Yunnan were used as controls. The full length HBV preS2/S and C genes were amplified by PCR, cloned, sequenced, and inserted into the prokaryotic expression vector p lambda PR. The recombinant plasmids p lambda PR-S2S and p lambda PR-C were transfected into E. coli of the line TOP10. The expression of the non-fusion proteins encoded by the HBV preS2S and C genes was determined by sodium dodecyl sulphate polyacrlamide gel electrophoresis (SDS-PAGE) and Western blotting. The antigenicity of the non-fusion proteins was examined by ELISA. Fifty samples of serum of patients with hepatitis A, 50 samples of serum of patients with hepatitis C, and 50 samples of serum of healthy blood donors were used as controls in the study of the antigenicity of non-fusion proteins. RESULTS: SDS-PAGE showed that the recombinant plasmids p lambda PR-S2S and p lambda PR-C were both stably and highly expressed in the E. coli for all 50 CHB patients. The molecular weights of the expressed non-fusion proteins, with a concentration of 16% and a purity of 50%, were between 31,000 and 21,000. Western blotting and ELISA showed that the purified recombinant non-fusion proteins reacted strongly with the antibodies HBpreS2S/SAb and HBcAb and the serum of CHB patients, but there was no cross-activity between the non-fusion proteins and all the serum samples of controls with HA and HC, and normal controls. CONCLUSION: The HBV preS2S and C genes from the minority nationality patients with CHB can be stably and highly expression in E. coli. The non-fusion proteins encoded by the HBV preS2S and C genes have high antigenicity. These findings have potential values in development of HB vaccines.

[Identification of hepatitis B virus genotypes in patients with chronic hepatitis B from different nationalities in ethnic minority areas in Yunnan Province, China].

OBJECTIVE: To determine whether there is evolutionary difference in hepatitis B virus (HBV) genotypes among the patients with chronic hepatitis B (CHB) of different nationalities and its clinical significance. METHODS: Peripheral blood samples were collected from 50 CHB patients, 25 of diverse nationalities and 25 of Han nationality from the ethnic minority regions in Yunnan Province, China, The HBV preS2/S (pre S2/S) and C genes were amplified by PCR. The PCR products were inserted into the vector pBluescriptIISK (pBS). The cloned preS2/S and C genes were sequenced. RESULTS: The sequences of HBV preS2/S and C genes from the 50 patients were 846 (with 49.1% of GC) and 552 (with 46.1% of GC) nucleotides (nt) in length, and encoded 281 and 183 amino acids (aa) respectively. These findings were registered in GenBank Accession Numbers: AY517619, AY517620, AY517488, AY517489, AY517598, AY517599. Compared with the HBV and subtype sequences in the GenBank database, the HBV preS2/S and C genes among all the subjects were homologous to ayw1 in sequence by 97.5% - 98.6% and 94.5% - 97.8% respectively. The "a" determinant region of S genes in all cases were found to be Arginine (AGA) and Lysine (AAA) at corresponding aa 122nd and 160th respectively. HBV genotype B was identified in all patients with CHB (ayw1 subtype). Genotypes A, C, D, E, F, G, and H were not detected in any of them. The quasi-species nature of the HBV in the sera was observed in 2 of the 50 samples examined (4%). There was not a significant difference in the prevalence of HBV genotype B between the 25 diverse nationality patients and the 25 control Han nationality patients (P > 0.05). In the 50 CHB patients, the preS2/S genes were identified to have aa substitutions at the positions R124K (1.1%), L172P (1.3%), M306T (1.5%), and I361M (1.6%), with a frequency of more than 1%. In all subjects, the frequency of aa G145R (0.4%) substitutions was less than 1%. In all subjects, nt variations of C genes caused aa substitutions among aa 27 - 63, 80 - 110, and 135 - 153 involved in T and B cell epitopes. In 45 CHB patients, C genes was identified to have aa substitutions at the positions V 27, N38, V63, Q135, and A147, due to nt variations of 1979A to G, 2012T to A, 2088G to T, 2304C to A, and 2339A to G transitions respectively. The frequency of aa substitutions of C genes was more than 1%. Whereas as for the other 5 severe CHB patients, the C gene variations of A to G, and A to C transitions at nt positions 2159 and 2189 led to aa substitutions of S to G and I to L at positions G87 and L97. No insertion or deletion was found in preS2/S and C regions. HBV genotype B was not relevant to different nationalities (all P > 0.05). CONCLUSION: It is the first time that the genotype of the HBV epidemic strains in the ethnic minority areas of Yunnan Province has been identified as genotype B subtype ayw1. The HBV genotype B is not related with nationality. A novel genotyping method by using PCR, gene cloning, followed by DNA sequencing that can identify all major genotypes has been developed. HBV genotype B is the geographic original strain in this area and is correlated with the severity of liver diseases and curative effect. HBV viral is the only significant variable associated with the CHB patients' prognosis.