We

We inhibitor price chose calendar time from 1 October 2009 as the time scale and we considered vaccination as a time varying covariate. Calendar time was chosen as the time scale for analysis to control for potential seasonal effects in incidences of the outcomes under study. In a second model we conditioned further on the number of in-hospital admissions and visits to specialist care one year before vaccination. The results are presented as hazard ratios with 95% confidence intervals, with 0.05 considered as the level of statistical significance. We estimated the number of cases attributable to vaccination in the vaccinated group as the number of observed cases in the vaccinated group multiplied by 1?1/hazard ratio. We estimated the absolute excess risk among vaccinated people as the number of cases attributable to vaccination divided by the number of vaccinated people.

The hazard ratios related to vaccination were stratified on time since vaccination and calendar time of vaccination. We used six weeks as a cut-off point for stratification according to time since vaccination. The early phase of the vaccination campaign was defined by vaccination within 45 days of 1 October 2009 and the late phase by vaccination after that period. The vaccinations started on 13 October (except for 10 people who had been vaccinated earlier); the cut-off point used includes the first 32 days of the vaccination campaign (43.6% of all vaccinated people) in the early phase. To investigate whether there was an acute effect of the vaccination (that is, non-proportional hazards on the time scale time since vaccination), we analysed estimates stratified according to both times (see table 4).

We used likelihood ratio tests to determine the interactions between calendar time of vaccination and time since vaccination. Post hoc analyses In an attempt to further estimate the influence of underlying comorbidity on health outcomes in patients undergoing H1N1 vaccination, we also estimated short term mortality according to vaccination status. Results By 31 March 2010 virtually all vaccination activity had been completed, with a cumulative 1024019 people vaccinated (52.6% of the study population; figurefigure).). In all, 222388 people, of whom 66.4% received a second dose, were vaccinated between the ages of six months and 12 years. Those belonging to a high risk group were largely over-represented compared with the total population during the first six weeks of the campaign (mid-October to November 2009); 74% of all those Drug_discovery vaccinated in the first week and 30% in the sixth week. In a second phase (from the beginning of December) the remainder of the population was offered vaccination.

All HCV treatment na?ve patients with a positive PCR were include

All HCV treatment na?ve patients with a positive PCR were included. Exclusion criteria included (1) pediatric patients; (2) chronic renal failure patients; (3) previous non-responders and those who had a relapse following http://www.selleckchem.com/products/CHIR-258.html prior treatment; (4) post renal transplant patients; and (5) patients with cirrhosis and signs of portal hypertension. Biochemical assessments, including ALT (normal value: 0-58 IU/L), AST (normal value: 0-36 IU/L), ��-glutamyltransferase (GGT), alkaline phosphatase (ALP), bilirubin, albumin, and coagulation profile, were done according to our laboratory standards. Serum HCV RNA was extracted using an automated extraction system. HCV detection and quantification were performed using the COBAS Ampliprep? /COBAS TaqMan? HCV test (Roche diagnostics) utilizing different sets of primers and probes, which target a conserved region of the 5�� untranslated region of the genome.

The main processes of this procedure include: (1) specimen preparation to isolate HCV RNA; (2) reverse transcription of the target RNA to generate complementary DNA (cDNA) and (3) simultaneous PCR amplification of target cDNA and detection of cleaved dual-labeled oligonucleotide detection probe specific to the target. This assay detects and quantifies HCV genotype (1-6) with a detection limit that ranges from 15 to 69 000 000 IU/mL. Prior to treatment, the HCV genotype was assayed using INNO-LiPA HCVII (Innogenetics, Ghent, Belgium). After reviewing the patients�� data, 240 patients fulfilling the inclusion criteria were selected for the study. Epidemiological data and treatment records were reviewed.

Liver biopsy is not done routinely in our center unless there is suspicion of underlying cirrhosis or a concomitant liver disease. For data entry, patients were divided according to their gender, viral load (HCV RNA below vs above 8 00 000 IU/mL), age (younger vs older than 40), treatment (PEG-IFN ��-2a vs PEG-IFN ��-2b), liver enzyme pattern (normal vs abnormal), and body mass index (BMI) (below vs above 28) (Table (Table11). Table 1 Patients�� characteristics Patients received either PEG-IFN ��-2a (40 Kd; PEGASYS, F. Hoffmann-La Roche, Basel, Switzerland) at a dose of 180 mg/wk or PEG-IFN ��-2b (12 Kd; PEGINTRON, Schering-Plough LTD, Singapore) at a dose of 1.5 mg/kg, and a standard dose of 1200 mg ribavirin with no weight related adjustments.

While on treatment, patients were followed monthly or more frequently if required. Complete blood count (CBC) and liver enzymes were checked at each visit. To maintain the starting dose of PEGylated interferon and ribavirin, erythropoietin and granulocyte-colony stimulating factor (G-CSF) were given as early as possible to overcome any potential treatment-induced anemia or neutropenia. Patients Cilengitide with genotype 2 and 3 were treated for 24 wk, while patients with genotypes 1, 4 and 5 were treated for 48 wk.

Neither of these genes appeared to respond to infection or to be

Neither of these genes appeared to respond to infection or to be differentially expressed, although Ptp4a1 selleck chemicals was highly expressed throughout the infection (not shown). Other Epo responsive genes did appear to respond to infection: Eif1a (Eukaryotic translation initiation factor) and Kif3a (kinesin family member 3A) were up regulated in all three mouse strains at day 7 and 9 respectively (Fig 6). However, since these genes participate in multiple signal transduction pathways, their up-regulation may be related to inflammation rather than erythropoiesis [32]. Figure 6 EPO responsive genes. Erythropoietin primarily acts through the erythropoietin receptor (Epor) that is exclusively expressed on cells from the erythroid lineage. So the level of expression of Epor may be related to the number of erythroid cells in the tissues.

In uninfected mice, Epor transcription was highest in A/J mice. After infection C57BL/6 mice tended to have lower levels of expression than either A/J or BALB/c mice although this was only significant if the expression levels were compared over the whole time course (p=0.00003 with respect to BALB/c mice and p=0.043 with respect to A/J mice). This was consistent with lower erythropoiesis and haemoglobin titre in C57BL/6 mice. However, given the small difference in expression and the lack of evidence for changes in Epo response genes this may not be an important mechanism driving anaemia after T. congolense infection. Interferon gamma (Ifng) down regulates Kit ligand (Kitl) and Epor and may be an important contributor to anaemia of infection [33].

Kit (CD117) is a receptor for Kitl (SCF or Stem Cell Factor), which acts synergistically with Epo in the promotion of erythropoeisis [33]. Ifng expression Batimastat increased approximately 8-fold after infection in all mouse strains and then declined fastest in BALB/c and remained highest in C57BL/6 consistent with the observed haemoglobin titres (Fig 7). Consistent with the inhibitory activity of IFN-��, the expression of Kit and Epor all declined somewhat at day 7 pi and C57BL/6 had the lowest levels of expression after day three (Fig 7). Higher levels of Kitl may be indicative of higher levels of erythropoiesis. In the spleen Kitl expression was highest in A/J and lowest in C57BL/6 from day 3 to 9 (Fig 7). Figure 7 Genes that mediate haematopoiesis. Insulin like growth factor (Igf1) appears to be more important than Epo for regulation of erythropoiesis in some anaemic patients [34], [35], [36]. Expression of Igf1 declined in C57BL/6 mice till day 7 pi, while it increased in A/J mice and was more than twice as high as in C57BL/6 on day 7 (Fig 7). However, differences at other days were small and no correlations with anaemia could be made.

Table 2 List of the 15 tumor-associated antigens used in the stud

Table 2 List of the 15 tumor-associated antigens used in the study. ELISA methodology ELISA was used to measure the humoral immune response in the serum or plasma of participating women to the various peptides or whole proteins antigens (Table 2). At each location, a specific standardized read FAQ ELISA protocol was followed (described below) on local samples to ensure assay consistency across sites. Each sample was given a barcode identifier in the laboratory to ensure a blinded analysis. White ��Maxiorp�� 96 wells plates (Nunc, Roskilde, Denmark) were coated with commercial antigens at concentrations ranging 2�C6 ��g/mL for proteins, and 0.25�C1 mg/mL for peptides in phosphate-buffered saline (PBS) and blocked with Well Champion reagent (Kem-En-Tec, Taastrup, Denmark) according to the manufacturer��s instructions.

Serum or plasma samples (100 ��L) were loaded in 6 serial dilutions starting at 1:40�C1:320 in 1% skim milk in PBS (Fluka, St. Louis, MO, USA) for each of the coated antigens in the plates and incubated at 37oC for 1.5 h with gentle agitation. The plates were washed 8 times with 300 ��L of Dulbecco��s PBS, 0.05% Tween 20 (PBST), and 1:10,000 horseradish peroxidase conjugated goat anti-human IgG (Chemicon, Temecula, CA, USA) was added for 1 h at 37oC, followed by 4 washes with 0.025% PBST. EZ-ECL (Biological Industries, Beit-Haemek, Israel) was used for luminescent development according to the manufacturer��s instructions. Luminescence was measured with Luminoscan Ascent (Thermo Scientific, Waltham, MA, USA) using Ascent software (Thermo Scientific).

Results were loaded into an internet database in a secure server according to the barcodes. Statistical methods All statistical analyses were performed using STATA 12 SE (StataCorp, College Station, TX, USA). All P-values were two-sided. P-values below 0.05 were considered significant. No corrections for multiple comparisons were performed. The initial data for each sample consisted of 6 measurements of AAb relative luminescence units (RLU) for each antigen in 6 consecutive dilutions. In the first step, we fitted the log10[RLU] as a linear function of the log10[dilution]. If the goodness of fit was not satisfactory, we excluded one outlier and refit the data by linear regression with the remaining 5 points. Reference values of the dilution were fixed for each AAb.

If the Dacomitinib goodness of fit was high, the fitted value at the reference point was calculated for each AAb. Otherwise the value was classified as ��missing�� for this antigen only, meaning that the data did not pass the quality control. A missing value was only given to a specific antigen and not to a sample. Thus, each sample was left with a set of maximum 15 values of AAb log10[RLU] for all antigens, each at the reference dilution point chosen for the antigens.

Next, the frequency of polymerase drug resistance mutations was a

Next, the frequency of polymerase drug resistance mutations was assessed in the study samples. The sequences of the polymerase gene derived from the patient samples Crenolanib mechanism were analyzed using the DRI, G2P, and STAN interpretation systems. The data revealed that in 22 of the 237 sequences (9%) polymerase mutations which are associated with drug resistance were present. The concordance of mutations identified as relevant for resistance between all interpretation tools was 100%. However, the interpretation of these mutations by the three algorithms differs. Where STAN just lists detected drug resistance-associated mutations, G2P and DRI rate them on a scale of three resistance ��levels�� (susceptible, possibly resistant, and resistant), and some mutations are assigned a different rating by the respective system.

When rating the mutations, G2P and DRI concurred in 9 of 22 cases. A detailed overview of the concordant and discordant results obtained by the two assays is presented in Table 1. Table 1 Sequence analysis results for resistance-associated mutations in the polymerase sequence (P) of samples and their rating as determined by DRI and G2P We further investigated whether and to what extent drug resistance mutations in P occur in the patient samples which potentially influence antibody binding and/or HBsAg secretion in the corresponding S sequence. Since neither of the interpretation algorithms includes these mutations, they were assessed according to previously published data (23).

The analysis of the 22 sequences with resistance mutations in P revealed that 19 of these sequences contained mutations which potentially influence antibody binding and/or HBsAg secretion in the corresponding S sequence, namely, pA181T, pM204I, and pM204V. Adding these sequences to those with MARABs as detected by the algorithms brings the total of sequences with mutations possibly influencing quantitative detection of HBsAg (MUPIQHs) to 72 (30% of all sequences analyzed) when using DRI and to 44 (19% of all sequences analyzed) when using G2P. An overview of the mutations in P detected in our sequences is shown in Table 1. Genotyping and assignment of sequences to patients’ country of origin. We then analyzed the HBV sequences for their genotype. Alignment and phylogenetic analysis using the respective P nucleotide sequence was performed, and the samples were analyzed by three interpretation systems.

The concordance of predicted genotypes between the three interpretation systems used was 100%. Overall, 37 samples were assigned to genotype A, 17 to genotype B, 23 to genotype C, 155 to genotype D, and 5 to genotype E. The patients’ country of origin could be determined by chart review in 186 cases. The association between country group of origin and genotype is presented in Fig. 2. Fig AV-951 2 HBV genotypes of the 237 individuals included in the study, grouped according to the patients’ region of origin.

This

This http://www.selleckchem.com/products/BI6727-Volasertib.html study was part of a prospective single-centre study on early enteral nutrition vs total parenteral nutrition in AP, where parts of the data on IL-6 and CRP have been published[29]. Venous blood was taken for measurement of plasma levels of TF, FVII, fibrinogen, IL-6 and CRP. Not all markers were measured at all time points in the study. TF and IL-6 were measured at inclusion, after 12 h, and after 1 and 3 d. CRP was measured at inclusion, and after 1 and 3 d. Fibrinogen and FVII were only measured at inclusion in the study. Descriptive data were recorded including age, gender, aetiology, time from onset of pain to inclusion in the study, Acute Physiology and Chronic Health Evaluation (APACHE) II score on day 1 and 3, organ failure, and mortality.

The severity of pancreatitis was assessed according to the Atlanta classification[30]. Blood samples and assays Peripheral blood samples were taken from each patient on study inclusion, at 12 h, and after 1 and 3 d. Admission plasma levels of FVII were analysed, and to detect the prevalence of fibrinolysis and fibrinogen consumption at admission, plasma fibrinogen was analysed. Fibrinogen is an acute phase protein, affected by pathologic proteolysis such as in disseminated intravascular coagulation, where low levels of fibrinogen are to be expected. TF, IL-6 and CRP were analysed at repeated time points during three days after inclusion in the study. Tissue factor and fibrinogen were collected using citrate tubes, and ethylenediaminetetraacetic acid tubes were used for IL-6 and CRP.

All samples were centrifuged at 2200 g for 10 min (3200 r/min, rotor diameter 19.1 cm). The plasma was decanted and stored at -70��C until further analysis. TF and FVII were assessed by enzyme-linked immunosorbent assay (ELISA)-kits according to the manufacturer��s instructions (Assaypro St. Charles, MO, USA). The TF-ELISA recognizes TF-apo, TF and TF-VII complexes. The FVII-ELISA detects free FVII and FVIIa, as well as complexes with TF, TF/factor VII and TF/FVIIa. Fibrinogen was analysed by Sysmex CA-7000 (Sysmex Corporation, Kobe, Japan) according to the operator��s manual. The procedure involves mixing citrate plasma with buffer. After incubation, coagulation was initiated by adding an excess of thrombin. The time between addition of thrombin and coagulation was registered photo-optically and is inversely proportional to the concentration of fibrinogen. IL-6 was measured by an ELISA-kit according to the manufacturer��s instructions (Quantikine, R6D systems Europe, Abingdon, UK). CRP was measured by Cobas 6000 (Roche Anacetrapib Corporation, Basel, Switzerland) according to the operator��s manual.

AZD929

LB42708? In bone marrow, the mean expression level of ID1 mRNA in stage IV (957��169) was significantly higher than other stages (P=0.003). Specifically, the levels of stages I, II and III were 54��185, 472��208, and 767��205, respectively. To confirm the specificity of ID1, we performed RT�CPCR analysis of six representative cases in each stage, which was very close to the average value (Figure 1C). In addition, sequencing of these transcripts confirmed that it was the product of ID1 (Supplementary Figure 1). Figure 1 The mean value of ID1 mRNA expression normalised GAPDH in bone marrow (A) and peripheral blood (B) according to staging classification. Group stage I consisted of patients with tumours that invaded less than the sub-mucosal layer and no lymph node metastasis …

Expression of ID1 mRNA in peripheral blood of gastric cancer In the peripheral blood samples, there was a significant relationship between the expression level of ID1 mRNA and the progression of gastric cancer cases (Figure 1B). The mean expression level of ID1 mRNA in stage IV (105��15) was significantly higher (P=0.0001) than stages I, II and III (12.4��15.4, 29.6��15.5, and 38.3��16.0, respectively). In addition, there was a significant correlation between the expression of ID1 mRNA in bone marrow and peripheral blood (r=0.23, P=0.002, data not shown). ID1 expression and clinicopathological features of gastric cancer patients We examined the clinicopathlogical significance of ID1 mRNA in samples from bone marrow and peripheral blood (Table 1).

In both bone marrow and peripheral blood, there are significant associations with many cliniopathlogical features such as tumour size and depth of tumour invasion. Especially, in patients with evidence of lymphatic invasion, lymph node metastasis or peritoneal dissemination, we found significantly higher expression of ID1 mRNA in bone marrow samples compared to patients without metastasis. (P=0.001, P=0.001 and P=0.002, respectively, Figures 2A�CC). Similarly, in peripheral blood samples, the cases with lymphatic invasion, lymph node metastasis or peritoneal dissemination had significantly higher expression of ID1 mRNA compared to patients without metastasis. (P=0.02, P=0.02 and P<0.0001 respectively, Figures 3A�CC). Figure 2 Comparison of the ID1 mRNA expression in bone marrow of patients with or without lymphatic invasion (ly) (+: n=156, ?: n= 104; A), lymph node metastasis (N) (+: n=188, ?: n= 79; B) and peritoneal cytology (cy) or peritoneal ... Figure 3 Comparison of the ID1 mRNA expression in peripheral blood of patients with or without lymphatic invasion (ly) (+: n=110, ?: n=68; A), lymph node metastasis (N) (+: n=132, -: n= 50; B) and peritoneal cytology (cy) or peritoneal Drug_discovery

If the exposure clustering accuracy rates decreased, the original

If the exposure clustering accuracy rates decreased, the original variables were retained. If not, the subset was removed from the model. This strategy maximized the information used to characterize exposure and minimized model complexity). Finally, we tested whether the graded exposure effect references was evident on the three fetal growth outcomes using s-FCM�Cderived exposure groups. Results Consistent with Fang et al. (2011) using the MIDS dataset, three exposure groups were identified, with an exposure accuracy rate of 94% and an inconsistency rate of 0%. Validation indices and graphs further supported three optimal number of latent classes (XBmi clearly revealed three optimal latent classes.

The other two indices also favored three classes, as their constantly increasing or decreasing properties showed only a minimal difference or trivial advantage at larger number of latent classes in comparison to three classes (see Supplementary Figure 1). Sammon mapping further supported three classes (see Supplementary Figure 2), where asterisks represent the projected centroids and dots represent subjects within the identified classes. The values on the two axes are the projected normalized scores for these subjects). Figure 1 shows the variation in self-reported smoking, urinary cotinine levels, and serum cotinine levels during pregnancy among the nonexposed (NE, N = 536), lighter-exposed (lTE, N = 229), and heavier-exposed (hTE, N = 213) groups. Similar to results with the MIDS dataset, the hTE group had the most intensive variation, followed by lTE and NE. Figure 1.

Functional curves of self-reported smoking (upper panel, x-axis: Trimester 1, 2, and 3), cotinine level in maternal urine samples (middle panel, x-axis: Trimester 1, 2, and 3) and in maternal serum (lower panel, x-axis: Trimester 1, 2, and 3) for the … Table 1 shows differences in exposure attributes between lighter- and heavier-exposed groups. For self-reported smoking (number of cigarettes per day, nicotine levels, and nicotine dependence items), the values reported for the s-FCM identified groups with MISSEB and MIDS were comparable but not Brefeldin_A for urinary cotinine levels. Table 1. Differences in Tobacco Exposure Attributes Between NE, lTE, and hTE neonates Also consistent with results with the MIDS dataset (Fang et al., 2011), the offspring of the hTE group weighed less at birth than those in the NE group, the estimated M difference was ?205.39 g (SE = 48.01, p < .001), and the lTE and NE groups were comparable in offspring birth weight (M difference = ?3.64 g, SE = 44.84, p = .94).

Anti-RR reactivity was detected in 41 patients

Anti-RR reactivity was detected in 41 patients thorough treated with interferon-�� plus ribavirin (38%), but in none of the patients treated with either one of the two drugs (p=0.0001; Fisher’s exact test, Figure 2). In contrast, other IIF-HEp-2 patterns were more frequent in patients receiving only interferon-�� (52.2%) than in those treated with interferon-�� plus ribavirin (25%) and those treated only with ribavirin (33.3%) (p=0.01) (Figure 2). Accurate information on treatment was available for 21 patients with HIV/HCV co-infection. Anti-RR reactivity was detected in 5 of the 20 patients treated with interferon-�� plus ribavirin (20%), but not in the single patient treated only with interferon-��. Figure 2 Antibodies to rods and rings are predominantly observed in HCV patients undergoing treatment with interferon-�� plus ribavirin.

The apparent association of anti-RR reactivity and therapy with interferon-�� plus ribavirin was further strengthened by the analysis of serial samples of RR-positive patients before and after the beginning of treatment. For this analysis, anti-RR reactivity was compared in samples obtained before and each month after the beginning of treatment, and also in samples obtained after treatment had been discontinued. As observed in Figure 3A, all pre-treatment samples were anti-RR negative, and an increasing frequency of anti-RR was observed during the several months of treatment. Even after discontinuation of therapy with interferon-�� plus ribavirin, some anti-RR reactivity was maintained when evaluated up to one year later (Figure 3A).

All time-point groups after initiation of treatment showed higher frequency of anti-RR reactivity compared to pre-treatment samples (p<0.001; Pearson Chi-square test). In contrast, there was no difference in the frequency of other IIF-HEp-2 patterns in samples obtained before and after initiation of therapy with interferon-�� plus ribavirin (Figure 3B, p=0.727). In addition, no anti-RR reactivity was observed in the serial samples from 27 untreated patients. Treated patients showed no statistical difference regarding the frequency of anti-RR during the months on treatment (Figure 3A, p=0.705). Figure 3 Anti-RR reactivity occurs at varying intervals after the beginning of treatment. There was consistent information on the profile of therapeutic response for 125 patients, for whom no association was observed between the appearance of anti-RR and therapeutic response. The proportion of non-responders was equivalent (p=0.271; Pearson Chi-square test) in the 39 patients with anti-RR reactivity (77%), 40 patients with other IIF-HEp-2 patterns (60%), and 46 patients with negative IIF-HEp-2 Drug_discovery tests (67%, Figure 4A and 4B).

Furthermore, an antigen-antibody chimera was reported to provide

Furthermore, an antigen-antibody chimera was reported to provide higher expression levels, better selleck compound yields, and increased stability in plant expression systems [2, 17]. In the present study, 3 different recombinant human colorectal cancer antigen GA733 genes were expressed in a tobacco (Nicotiana tabacum) plant expression system: GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), GA733-Fc with KDEL (GA733-FcK), and GA733 with KDEL (GA733K). The stability and functionality of these colorectal cancer vaccine candidates were confirmed by western blot analysis and ELISA, respectively. In order to understand the fusion of Fc to GA733 and its functionality, the immunogenicity of recombinant GA733-Fc with oligomannose glycosylation was investigated in mice. 2. Materials and Methods 2.1.

Construction of the Plant Expression Vector The synthetic DNA sequence encoding GA733 (Gln38-Lys279, GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY189981″,”term_id”:”28864237″,”term_text”:”AY189981″AY189981) was modified by N-terminal extension with a 30-aa plant ER signal peptide (MATQRRANPSSLHLITVFSLLAAVVSAEVD) from Nicotiana plumbaginifolia and C-terminal extension with an ER retention signal (KDEL). The recombinant chimeric protein GA733-Fc was generated by fusing GA733 to the Fc fragment of human IgG1 (Val97-Gly328, GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY172957″,”term_id”:”27728676″,”term_text”:”AY172957″AY172957). The Lys279 (C-terminus of GA733) was followed by the Val97 (N-terminus of Fc fragment of human IgG1).

The GA733-Fc-encoding sequences were placed under the control of the enhanced cauliflower mosaic virus (CaMV) 35S promoter and tobacco etch viral 5��-leader sequence (TEV). The GA733, GA733-Fc, and GA733-FcK expression cassettes were subcloned into the HindIII sites of the binary plant transformation vector pBIN-Plus to yield pBI GA733K, pBI GA733-FcK, and PBI GA733-Fc, respectively (Figure 1(a)). Figure 1 Schematic diagram of plant expression vectors and recombinant proteins. (a) Gene expression cassettes of GA733K, GA733-FcK, and GA733-Fc. E/35S-P, the Cauliflower mosaic virus 35S promoter with duplicated enhancer region; TEV, untranslated leader sequence … 2.2. Plant Transformation Recombinant vectors were introduced into the Agrobacterium tumefaciens strain LBA4404 by electroporation.

Transgenic GSK-3 tobacco plants were generated by Agrobacterium-mediated transformation [11]. Transgenic tobacco lines were selected on Murashige and Skoog medium (Dachfu, Haarlem, Netherland) containing 100mg/L kanamycin. Transgenic plantlets were then transferred to soil in a growth chamber at a constant temperature of 23��C and 70% humidity and were maintained under a 14:10h light-dark cycle. Transgenic and nontransgenic N. tabacum plants were grown in a greenhouse under controlled conditions. 2.3.