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In this paper, using capillary zone electrophoresis method determined gallic acid content in Paeonia lactiflora Pall. Under the condition of buffer solution containing 20 mmol/L Na2B4O7, effective separation of components to be tested was realized. Measured content of gallic acid in Paeonia lactiflora Pall was 1.15 mg/g (RSD=4.28%) (n=6), the average recovery was 88.48% (n=5). This method is simple and fast.

In this paper, using capillary zone electrophoresis method determined gallic acid content in terminalia. Under the condition of buffer solution containing 20 mmol/L Na2B4O7, effective separation of components to be tested was realized. Measured content of gallic acid in terminalia was 25.36 mg/g (RSD=6.8%) (n=6), the average recovery was 87.6%. This method is simple and fast.

The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats.

A novel antiviral protein was purified from an extract of Grifola frondosa fruiting bodies using a procedure that included 40% ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography, and designated GFAHP. This protein inhibited herpes simplex virus type 1 (HSV-1) replication in vitro with an IC(50) value of 4.1 microg/ml and a therapeutic index >29.3. Higher concentrations of GFAHP (125 and 500 microg/ml) also significantly reduced the severity of HSV-1 induced blepharitis, neovascularization, and stromal keratitis in a murine model. Topical administration of GFAHP to the mouse cornea resulted in a significant decrease in virus production (mean virus yields: 3.4log10PFU in the treated group and 4.19log10PFU in the control group). We proved that GFAHP directly inactivates HSV-1 while simultaneously inhibiting HSV-1 penetration into Vero cells. Gel electrophoresis showed that GFAHP had a molecular weight of 29.5 kDa. GFAHP was tryptic digested and analyzed from the PMF of matrix assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and nanoelectrospray ionization tandem mass spectrometry. The N-terminal sequence of GFAHP consisted of an 11 amino acid peptide, NH(2)-REQDNAPCGLN-COOH that did not match any known amino acid sequences, indicating that GFAHP is likely to be a novel antivirus protein. To our knowledge, this is the first report that characterizes an anti-HSV protein from G. frondosa.

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (

In this study, the persistence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was observed in feces, urine and water. In addition, the inactivation of SARS-CoV in wastewater with sodium hypochlorite and chlorine dioxide was also studied. In vitro experiments demonstrated that the virus could only persist for 2 days in hospital wastewater, domestic sewage and dechlorinated tap water, while 3 days in feces, 14 days in PBS and 17 days in urine at 20 degrees C. However, at 4 degrees C, the SARS-CoV could persist for 14 days in wastewater and at least 17 days in feces or urine. SARS-CoV is more susceptible to disinfectants than Escherichia coli and f2 phage. Free chlorine was found to inactivate SARS-CoV better than chlorine dioxide. Free residue chlorine over 0.5 mg/L for chlorine or 2.19 mg/L for chlorine dioxide in wastewater ensures complete inactivation of SARS-CoV while it does not inactivate completely E. coli and f2 phage.

The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5′ nontranslated regions (5′NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5′NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5′NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.

Ceratoides lanata is the native sand-fixing plant in deserts of America Continent. As an even-green undershrub with extremely strong antifreeze capability, Ceratoides lanata had been considered as the winterfat and introduced into many countries including China. To explore the mechanism of cold resistance and to clone genes directly involving the strong antifreeze capability, a normalized cold-stressed full-length cDNA library was constructed from the cold-treated leaves of Ceratoides lanata by the method of DSN (duplex-specific nuclease)-based normalization and SMART (switching mechanism at 5' end of RNA transcript) full-length cDNA construction. The quality check showed that the titer of un-amplified cDNA library was about 1.4×106 cfu mL−1 and the average size of cDNA inserts was about 1 150 bp with a recombination rate of 100%. The result of sequencing 187 clones selected randomly revealed that the redundancy of the cold-stressed normalized full-length cDNA library is only 0.59%, and the ratio of full length is 46.3%. Among 187 cDNAs, 24 have homology with known genes or proteins, while the other 163 have no identity, suggesting that most cDNAs are new cold-involved genes from Ceratoides lanata. The analytic result showed that the cold-stressed normalized full-length cDNA library of Ceratoides lanata a was established successfully, and this will benefit the further study on the molecular mechanism of strong antifreeze capability and cloning of cold-resistant genes

As a high stress resistance psammophyte with the characteristic of sand fixation and winterfat, Ceratoides lanata had been successfully introduced into many countries in recent years. The leaves and twigs of Ceratoides lanata in room temperature were used as the Driver and those treated by cold stress were used as Tester. Tester and Driver ds cDNA were prepared from their high quality mRNA which were purified from their total RNA. Tester and Driver cDNA were separately digested to obtain shorter, blunt-ended molecules by RasI. Then two Tester populations are created with different adaptors, while Driver cDNA has no adaptors. Differentially expressed genes were equalized and enriched by two round subtractive hybridizations using excess Driver population as compared with Tester population. The differentially expressed cDNAs were exponentially amplified by first round suppression PCR using the diluted hybridization product as template, and then the secondary PCR was performed using the first PCR product as template by nested primers to finally enrich the differentially expressed cDNAs, which consists of the SSH library of Ceratoides lanata. These cDNAs were inserted into vectors and 362 cDNA clones were obtained. The sequencing results showed that some cDNAs of cold-stressed Ceratoides lanata SSH library have relatively high homology with known stress resistance-related genes or proteins, and other cDNAs are new genes which are firstly reported here. Our result lays a foundation for the cDNA cloning of valuable genes including antifreeze genes, heat-resistant genes, drought-tolerant genes, alkali-salt-tolerant genes from Ceratoides lanata, and their transgenic application

In this study, D-fraction extracted from Grifola frondosa (GF-D) and its combination with human interferon alpha-2b (IFN) were investigated for the inhibitory effect on hepatitis B virus (HBV) in HepG2 2.2.15 cells (2.2.15 cells). HBV DNA and viral antigens were analyzed by a quantitative real-time polymerase chain reaction and end-point titration in radioimmunoassays, respectively. The results showed that GF-D or IFN alone could inhibit HBV DNA in 2.2.15 cells with the 50% inhibitory concentration (IC50) of 0.59 mg/ml and 1399 IU/ml, respectively. We further investigated the combination of GF-D and IFN for anti-HBV activity and found that they synergistically inhibited HBV replication in 2.2.15 cells. In combination with 0.45 mg/ml GF-D, the apparent IC50 value for IFN was 154 IU/ml. This 9-fold increase in antiviral activity of IFN suggested that GF-D could synergize with IFN. These results indicate that GF-D, in combination with IFN, might provide a potentially effective therapy against chronic HBV infections.

The transmission of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is associated with close contact to SARS patients and droplet secretions of those patients. The finding of positive RT-PCR results from stools of SARS patients suggests that stools of SARS patients or sewage containing stools of patients could transmit SARS-CoV. We used a novel style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing. We also used cell culture, RT-PCR and gene sequencing to detect and identify the viruses from sewage. No infectious SARS-CoV contamination was found in any of the samples collected, but the nucleic acid of SARS-CoV could be detected in the sewage from the two hospitals before disinfection. While the RNA was only detected in three samples from the 309th Hospital, the others were negative after disinfection. These findings provide strong evidence that SARS-CoV can be excreted through the stool/urine of patients into sewage system, thus making the sewage system a possible route of transmission.

In this study, to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine dioxide, cell culture, enzyme-linked immunosorbent assay (ELISA), and long-overlapping RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV before and after disinfection. The results revealed the complete inactivation of infectivity after a 10-min exposure to 7.5mg of chlorine dioxide per liter; and the highest level of sensitivity in the 5'non-translated regions (5'NTR) (the sequence from bp 1 to 671), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine dioxide; the complete destruction of antigenicity after a 10-min exposure to 7.5mg of chlorine dioxide per liter. It is suggested that the inactivation mechanism of HAV by chlorine dioxide was due to the loss of the 5'NTR and/or destruction of the antigenicity, which is not similar to that of chlorine (Appl Environ Microbiol 68: 4951).

To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV).A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-,671-, 1084-, and 1128bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV.All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus,21 PFU for Coxsackie virus,60 PFU for Echovirus and 105 TCID(50) for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID(50) for HAV.The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness.

This fiber optic sensor, based on competitive immunoreactions between coating-haptens and free haptens in solutions with corresponding antibodies, was developed to determine the concentration of the free hapten, methsulfuron-methyl. The ovalbumin (OVA)-methsulfuron-methyl conjugate was immobilized on a microscope slide. Horseradish peroxidase (HRP) labeled goat anti-rabbit IgG was used to generate an optical signal. The portable optical device consisted of a 0.25-W tungsten-halogen light source and a photosensitive diode detector. A typical competitive-binding calibration curve was seen between 0.3 and 100 ng/ml of methsulfuron-methyl. The detection limit for methsulfuron-methyl was 0.1 ng/ml.

A new type of electropositive filter media particle was tested to adsorb bacteriophage f 2 and enteric viruses from tap water. 3×nutrient broth (pH 7.2) was used to elute the adsorbed viruses, and the eluate was reconcentrated by polyethylene glycol ( M W 6000) precipitation with a final concentration of 10% (wt./vol.). The adsorption of bacteriophage was reliable and efficient, and not affected by the pH value, temperature, turbidity and organic materials in water. This method gave a recovery of Polio 1 virus 96.0% for small-volume tap water; 88.7% for large-volume water; and gave a comparable recovery of HAV, Coxsackie B 3 and Echo 7 from tap water. The concentration method need not acidify virus-containing water, add exogenous multivalent cation salts, or require expensive equipment.

To detect the common intestinal pathogenic bacteria quickly and accurately.A rapid (3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays.One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified.Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost.