, 2003). This reset was often accompanied by a difference in mean phases between the two stimuli,

shedding light on potential mechanisms for encoding and retrieval (Rizzuto et al., 2006). Phase resetting has also been seen in response to auditory stimuli (Lakatos et al., 2013 and Ng et al., 2013). On the other hand, there have been indications that the event-related potential generated by visual stimuli is due mainly to additive evoked potentials (Rousselet et al., 2007 and Shah et al., 2004). In studying mechanisms of behavioral responses, such as phase resetting and additive evoked potentials, a large number of variations are possible (Krieg et al., 2011 and Yeung et al., 2007). We have chosen to focus on the simple definition of phase resetting set forth by Shah et al. (2004): the response is characterized by an increase in coherence with no associated increase selleck screening library in CB-839 mouse power, and an ongoing oscillation is present before the stimulus. However, while the definition is simple, identification of a mechanism such as phase resetting requires the somewhat arbitrary selection of several criteria. We can measure

changes in power using a statistical test, but what significance level is appropriate? Should the change in power be measured relative to baseline values or relative to the prestimulus time period? In the case of the IPC, we can again use a statistical test (such as a Rayleigh test of uniformity) to identify time periods of increased phase coherence. However, we must still choose a significance level for the test. For example, an IPC of 0.15 may be statistically higher than chance at some p value, but visual inspection of the data will give no indication that a phase reset is occurring. Calculating the correlation between IPC and mean amplitude will bypass the need to choose these significance levels, but it may place too high of a value on small

deviations from the baseline. through Given that each electrode will have differing amounts of activity across the power spectrum that can obscure the oscillation of interest (here, at 2 Hz), we make the assumption that this added noise will lead to smaller changes in amplitude and IPC than we might expect. In other words, an IPC of 0.15 may not be valuable on its own, but its contribution to a larger distribution of points may allow for identification of the underlying mechanism. We therefore introduced a technique that uses the wavelet amplitude relative to baseline and the IPC, both measured at the peak of the response. Due to the variation in noise across electrodes, it produces a distribution of points for each brain region, and the shape and location of that distribution indicates which mechanism generated the response.

This is accomplished by increasing the concentration of acetylcholine through reversible inhibition of its hydrolysis by acetylcholinesterase. The recommended

initial dose of donepezil is 5 mg taken once daily. Donepezil is well absorbed with a relative oral bioavailability of 100% and reaches peak plasma concentrations (Cmax) approximately 3–4 h selleck compound after dose administration. In humans, donepezil is metabolized mainly by the hepatic cytochrome P-450 2D6 and 3A4 isozymes. 2 Elimination of donepezil from the blood is characterized by a dose independent elimination half-life of about 70 h. 3 and 4 Because plasma donepezil concentrations are related linearly to acetylcholinesterase inhibition, 5 plasma donepezil concentration is a useful tool to predict donepezil efficacy. In the literature, methods have been reported for the quantification of donepezil in biological fluids. Methods are reported for the quantification of donepezil from biological

matrix using high-performance liquid chromatography (HPLC) equipped with an ultraviolet detector,2 and 3 fluorescence detector4 and mass spectrometric1, 6 and 7 detector. Methods are also reported for the quantification of enantiomers of donepezil from human plasma.8, 9 and 10 Other methods are reported with estimation of donepezil in plasma by capillary electrophoresis,11 hydrophilic interaction chromatography-tandem mass spectrometry,12 direct measurement,13 automated Mephenoxalone extraction.14 The HPLC methods used to determine donepezil in human plasma are insensitive because

of the lower limit of quantification (LOQ of >1.0 ng/ml). Some of the Y 27632 reported methods1, 4, 6, 10, 13 and 14 utilized analogue internal standards like diphenhydramine, lidocaine, pindolol, loratadine, escitalopram, etc. and are validated with different calibration curve ranges for the estimation of donepezil from rat plasma, human plasma and other biological fluids. Usage of labelled internal standards is recommended during the estimation of compounds from the biological matrices to minimize the matrix effects associated with the mass spectrometric detection. Bioequivalence and/or pharmacokinetic studies become an integral part of generic drug applications and a simple, sensitive, reproducible validated bioanalytical method should be used for the quantification of intended analyte. Bioequivalence studies for the donepezil needs to be performed with the dosage of 10 mg and 23 mg tablets to support the generic abbreviated new drug applications. For the pharmacokinetic and bioequivalence studies, quantification of donepezil was sufficient and quantification of its metabolites shall not be required. During the bioequivalence studies, appropriate lower limit of quantification needs to be used to appropriately characterize the concentration profile including the elimination phase.

Help from specific CD4+ subsets of T cells to B cells is a prerequisite for this humoral immunity. Follicular T helper (TfH) cells are a newly recognized lineage of CD4+ T cells [11], that were

originally discovered in the B cell follicles of secondary lymphoid organs with the defining feature of high expression of the chemokine receptor CXCR5. There are accumulating evidences that these TfH cells are the key T-cell subset required for the formation of germinal centers (GCs) and the generation of antigen specific T cell-dependent antibody responses [11], [12], [13], [14] and [15]. That TfH cells are actively engaged in responses ABT-737 order to vaccination has been shown in a number of different virus systems. Bentebibel et al. reported that peripheral TfH-like cells, marked as CD4+ICOS+CXCR3+CXCR5+, are associated with protective antibody responses after seasonal flu vaccination [16]. The efficacy of the foot and mouth disease vaccine (FMDV) may also be enhanced through the generation

of TfH cells [17] and [18]. Furthermore, the non-responsiveness of HIV-infected individuals to the 2009 H1N1 vaccine has been primarily attributed to the impairment of circulating TfH cells [19]. In the case of HBV, the abnormal expressions of TfH-related molecules have been reported to be at least Ibrutinib ic50 partially responsible for the dysfunction of immune responses during chronic HBV infection [20] and [21]. Despite this clear evidence that TfH cells have an important role

in the humoral immune response to a number of vaccines, the relationship between TfH cells and specific antibody responses to HBV vaccine has not as yet received sufficient attention. Given the growing recognition of the importance of TfH cells in generating a strong humoral immune response, it seems reasonable to hypothesize that polymorphisms of TfH related molecules may be associated with non-responsiveness to HBV vaccination. Therefore, in this study a total of 24 single nucleotide polymorphisms (SNPs) within six genes (CXCR5, ICOS, CXCL13, IL-21, BCL6 and CD40L) were selected and analyzed. The cohort recruited for the current study was a subset from a previous survey Adenosine on non-responders to HBV vaccine [4] and [22]. The details for screening were described in Supplementary Fig. 1. In brief, a total of 37,221 ethnic Han Chinese volunteers with no hepatitis B vaccination history were recruited. All recruited volunteers were vaccinated with 10 μg of recombinant HBV vaccine (Shenzhen Kangtai Biological Products Co., Ltd., Shenzhen, Guangdong) according to the standard 0, 1, and 6 months vaccination schedule. Anti-HBs titers were tested at 7th month after initiating the vaccination regime and individuals whose anti-HBs titer was lower than 10 mIU/ml were re-vaccinated with a further 3 doses of HBV. Levels of Anti-HBs antibody were re-tested approximately one month after the final dose of vaccine was administered.

The Phase I, double-blind, randomized study in 50 healthy adults aged 18–49 years (CTRI/2010/091/000082) compared Ferroptosis tumor the safety of two Al(OH)3 adjuvanted whole virion formulations (10 μg and 15 μg haemagglutinin (HA) per dose). Satisfactory

42-day follow-up data led to authorization for the Phase II/III double-blind, randomized study, carried out in 330 individuals (110 adults, 110 elderly and 110 adolescents and children ≥3 years) at five sites in India (CTRI/2010/091/000093). Following single dose of either 10 μg or 15 μg HA of the study vaccine given intramuscularly at 21 days apart, safety and immunogenicity were assessed and the vaccine found safe in all age groups. After 42 days of follow-up, no SAEs were reported and none of the few unsolicited events detected in each group was causally related to the study products. All solicited reactions reported in the groups were similar, mild in intensity and resolved without sequelae. Immunogenicity was assessed on Day click here 0 and 21 by

HAI assay. The vaccine-induced immune responses of both formulations were in line with published studies [6], [7] and [8]. Seroconversion and seroprotection (HI titres ≥1:40) rates met the requirements of the European Medicines Agency (EMEA) and the US Food and Drug Administration (FDA) for licensure in all three age groups. The DCG(I) granted the licence to market the 15 μg adjuvanted vaccine on 6 August 2010. As soon as six months of stability data are available, the 10 μg formulation will be registered and launched under the brand name Enzavac® in India. All the clinical studies were approved by the DCG(I), the Independent Review Board and the Institutional Ethics Committee. Additionally, all data were periodically reviewed and approved by an independent Data Safety Monitoring Board. Among other controls, an on-site regulatory audit for the manufacturing processes and quality control Oxygenase testing was carried out by an inspection team from WHO, the CDSCO/DCG(I), and local FDA in April 2010. During the entire clinical development and licensing of the IIV and

LAIV, SII was actively supported by the government agencies since the need for a pandemic vaccine in India was clear. As a result, they approved importation of the H1N1 vaccine strain, clinical trial protocols and finally licensure on a fast-track basis. In parallel, SII proactively apprised these agencies of developments at each stage of the project. For instance, while requirements for the production and use of IIV are long established, the WHO guidelines for the manufacture and evaluation of LAIV were being updated. Policy-makers and the scientific community were also apprehensive over issues such as potential reversion of attenuation to virulent phenotype, emergence of more pathogenic viruses from reassortant between vaccine strain and wild type strain, and limited safety data.

001) and 65% versus 39% (P < 0.001), respectively.

Among placebo recipients, IgA response rates were generally comparable for subjects with and without a HAI response: 22% versus 30% for A/H1N1 (P = 0.5), 41% versus 28% for A/H3N2 (P = 0.2), and 31% versus 34% for B (P = 0.8). In year 2, 360 placebo recipients and 633 LAIV recipients had data for both HAI and IgA responses. For A/H1N1, A/H3N2 and B, HAI responses were 48% versus 16% (P < 0.001), 42% versus 16% (P < 0.001), and 29% versus 10% (P < 0.001) for LAIV versus placebo recipients, respectively. For LAIV recipients, IgA responses to A/H1N1, A/H3N2, and B were observed among 48% versus 35% (P < 0.001), 51% versus 38% (P < 0.001)

and 48% versus 36% (P < 0.001) of those with and without a HAI response, respectively. As in year 1, IgA responses among placebo recipients EGFR signaling pathway were generally comparable for subjects with and without a HAI response: 21% versus 33% for A/H1N1 (P = 0.1), 26% versus 28% for A/H3N2 (P = 0.9), and 42% versus 27% for B (P = 0.1). Selleckchem PD-332991 Based on pooled data from all 3 studies, in years 1 and 2, the mean postvaccination strain-specific to total IgA ratio was 3.1-fold higher (P < 0.01) and 2.0-fold higher (P = 0.03) among LAIV recipients with no culture-confirmed influenza illness compared with LAIV recipients who developed culture-confirmed influenza illness ( Table 3). For each individual study and each type/subtype, mean postvaccination IgA ratios were generally higher among LAIV recipients with no evidence of influenza illness,

although no individual comparison reached statistical significance. When the analysis was restricted to culture-confirmed illness due to vaccine-matched strains, a 3.0-fold difference in IgA ratios between those with and without illness was still present among LAIV recipients in year 1 (P = 0.02). However, in year 2, there were very few subjects who developed vaccine-matched influenza illness (N = 13); Phosphatidylinositol diacylglycerol-lyase the IgA ratio was 1.4-fold higher among those without influenza illness but this difference was not statistically significant (P = 0.59). In year 2 of study 3, there was a high incidence of influenza illness due to antigenically mismatched influenza B strains, due to significant circulation of viruses from the influenza B lineage not included in the vaccine; the B/Yamagata lineage strain B/Victoria/504/2000 was included in the vaccine but B/Hong Kong/1351/2002-like viruses of the B/Victoria lineage circulated. In year 2 of study 3, the mean IgA ratio against the vaccine-matched influenza B antigen was 1.8-fold higher among those subjects without illness compared with those with illness due to opposite lineage B strains (P = 0.15).

In addition, to assess Ag-specific Th cell responses, IL-6, IL-17, and TGF-β were measured in cell supernatants from lymphocytes restimulated with F1- and V-Ag by sandwich ELISA, as were IFN-γ and IL-10 (Fig. 8B). Although TGF-β was not detected (data not shown), Ag-specific IL-6 and IL-17 production was enhanced significantly, as well as IFN-γ and IL-10. For the i.m. immunization study, lymphocytes from spleens, HNLNs, and PLNs, which were obtained from each two DNA-vaccinated mice at 14 wks, were restimulated with F1-Ag, V-Ag, or media for 2 days (Fig. 9A). I.m. LTN DNA immunization also showed significantly Screening Library research buy Ag-specific enhancement of IFN-γ production, as well as IL-4, IL-5, and IL-10

in both spleens and LNs. In addition, IFN-γ, IL-6, IL-10, IL-17, and TGF-β were also measured in cell supernatants from lymphocytes restimulated with F1- and V-Ag by sandwich ELISA (Fig. 9B). Although TGF-β were not detected (data not shown), Ag-specific IL-6 and IL-17 production was enhanced significantly, as well as IFN-γ and

IL-10. These results suggest that both LTN DNA vaccines primed for Ag-specific T cells, and Th1-, Th2-, and Th17-type cytokines in the i.n.- and i.m.-immunized mice. In this study, to obtain an effective DNA vaccine against pneumonic plague, two DNA vaccines were constructed co-expressing the V-Ag or F1-V fusion protein in combination Tariquidar mouse with LTN DNA as a molecular adjuvant. Since Y. pestis is a facultative intracellular pathogen, Parent and co-workers suggested that plague vaccines should be designed to maximally prime both cellular and humoral immunity for

effective protection [13], [14] and [15]. LTN was selected as a molecular adjuvant because past studies have shown that LTN exhibits both Th1- and Th2-type properties when applied mucosally and parenterally [18], [19], [20], [21], [22], [23] and [24]. LTN is produced by CD8+ T cells, NK cells, and γδ TCR+ IEL, indicating induction of protection immunity against tumors through chemotaxis of T cells and natural killer (NK) cells [32] and [33]. LTN has also been adapted as a molecular adjuvant for development of vaccines against pathogens, including human immunodeficiency virus (HIV) [34] Ergoloid and avian coccidiosis [35]. For the development of an effective plague vaccine, we tested LTN as a molecular adjuvant against Y. pestis. In this study, the mucosal adjuvant effect by LTN to stimulate protective immunity was not as apparent when given nasally. Although nasal immunization with LTN/βgal DNA vaccine plus F1-Ag did appear to confer improved protection against pneumonic plague challenge, this was not significantly different from any of the vaccinated groups. Likewise, for i.m. DNA-vaccinated mice, protection conferred by the LTN/βgal DNA vaccine was not significantly different from the LTN/V or LTN/F1-V immunized mice. However, these results show that i.m.

Within each pair of twins, Dose 1 and Dose 2 of HRV vaccine/placebo was administered on the same day. In view of providing CFTR activator benefit to the infants receiving placebo during the course of the study, an additional dose of HRV vaccine was administered to all infants (aged < 6 months) at 7-weeks after the second vaccine/placebo dose in an open-labeled manner. All infants received three doses of combined diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus and Haemophilus influenzae

vaccine (DTPa-HBV-IPV-Hib [Infanrix hexa™, GSK Biologicals]). Infants were not allowed to take part in the study if they had received any investigational drug or vaccine 30 days preceding the first study vaccine/placebo dose or had a history of allergic disease likely to be exacerbated by the vaccine or had a history of chronic gastrointestinal diseases. They were also excluded if they were immunosuppressed or had an acute disease at the time of study enrolment. Hypersensitivity

to the vaccine/placebo and intussusception were adverse events that established absolute contraindication to further administration of vaccine/placebo doses. This study was conducted between January 2007 and February 2008, following Good Clinical Practice and the Declaration of Helsinki; the protocol and related documents were reviewed and approved by the ethics committee of the study centers. Parents or guardians of the participating twins provided consent for study participation by signing heptaminol the informed consent form. Rotarix™ (HRV) vaccine contained at least 106.0 median cell culture infectious dose of the Dinaciclib vaccine strain per vaccine dose (1 ml). The placebo had the same constituents as the active vaccine but without the vaccine virus and was identical in appearance to the vaccine. The lyophilized vaccine and placebo were reconstituted with the supplied liquid calcium carbonate buffer before oral administration [10]. Presence of the vaccine strain in the placebo group for any of the stool samples collected at pre-determined time points

was considered a positive transmission case. To evaluate rotavirus antigen shedding (ELISA, Dr. Ward’s Lab, USA), stool samples were collected by the parents/guardians in each pair of twins (HRV vaccine/placebo) at pre-determined time points—before the administration of the first and second HRV vaccine/placebo dose (or on the day of vaccination), three times a week (every two days) up to six weeks after each dose of HRV vaccine/placebo and at the post-vaccination blood sampling time point (7 weeks post-Dose 2). To ensure proper stool sample collection, surveillance was performed by a social worker at the time of stool sample collection. The study staff stuck appropriate labels on the stool collection containers to avoid mix-up of samples by the parents/guardians.

The median overall survival of the vaccinated patients was 19.2 months, calculated from the day of leukapheresis instead of from diagnosis of metastasis, as is done in unselected case series. Overall Alectinib mouse survival from date of diagnosis of metastatic disease in our dendritic cell vaccinated patients was 30.3 months. According to the American Joint Committee on Cancer Staging Manual, median overall survival is 17 months for M1a, 9 months for M1b, and 4.5 months for M1c.43 Our patients showed a median overall survival of 29 months for M1a, 22.5 months for M1b, and 6 months for M1c. No large difference in overall survival was seen in patients who received prior therapy for metastatic disease to treatment-naïve

patients. Comparing our results on survival with other published series, the observed median overall

survival of 19.2 months in dendritic Fludarabine cell-vaccinated patients not only exceeded the overall survival as reported in studies using systemic treatment (range, 5.2 to 15.3 months), but also the overall survival in almost all studies in more selected metastatic uveal melanoma patients treated with local therapies of the liver (range, 5.2 to 24 months), such as surgical resection of liver metastasis, hepatic artery chemoembolization, and hepatic artery infusion chemotherapy.17 These invasive therapies excluded patients with extrahepatic metastasis and high World Health Organization performance status, that is, have more strict inclusion criteria, and consequently included patients with more favorable prognostic factors. Further comparison with

a cohort of patients with a similar proportion of pretreated patients (12 of 20 patients) and selection criteria, treated with treosulfan and gemcitabine, showed a similar median overall survival (19.2 vs 17 months).44 Although our results do not allow definite conclusions about clinical outcome, the immunologic responses, previously shown to correlate with clinical outcome,28 and the observed long overall survival in our cohort of metastatic uveal melanoma patients seem promising. Additionally, the minimal toxicity associated Ketanserin with dendritic cell vaccination compares favorably with other treatment methods. As to metastatic patients, the high tumor burden may hamper the induction of effective immune responses, creating a suppressive tumor microenvironment by the secretion of suppressive cytokines and attraction of regulatory T cells.45 Robust immunologic responses on dendritic cell vaccination are induced more frequently in patients with no evidence of disease (72%) (manuscript in preparation) compared with patients with macroscopic tumor burden (32%).28 On the basis of the association of tumor-specific T cells and improved clinical outcome, this suggests that dendritic cell-based vaccination may have a more pronounced role in an adjuvant setting and should be initiated at an early stage after tumor resection.

Other less commonly used tests include the 2-km walk time with values ranging from 16.9 to 18.9 minutes, Docetaxel clinical trial and Rockport 1-mile test (reported values of 17.45 and 17.65 minutes). There were no published norms identified for the 12MWT, 2-km walk test or Rockport 1-mile test. Grip strength was the most commonly used upper extremity function test; it was used in 26 studies (see Table 3 in the eAddenda). The mean of the grip strength data

that could be pooled was 24.6 kg (95% CI 23.7 to 25.5) in women on treatment and 22.8 kg (95% CI 20.6 to 25.1) in women off treatment (Figure 7). These values fall below the median reported values of 27.7 kg for healthy adults aged 50 to 59 (Table 2).29 No heterogeneity was identified (I2 values < 20%). 1RM using a bench or chest press protocol was estimated in four studies and measured directly in four studies. The pooled mean for bilateral bench press 1RM was 20.9 kg (95% CI 17.0 to 24.7) in women on treatment and 23.9 kg (95% CI 21.0 to 26.8) in women off treatment (Figure 8). Moderate heterogeneity was identified (I2 = 36%) for women off treatment. Normative values for 1RM are reported in weight pushed per kg of body weight, but for a woman weighing 70 kg, these pooled values fall into the ‘very poor’ category across all age groups ( Table 2). 11 Other

methods of assessing upper extremity strength include a bench press 6RM, bench press endurance with various protocols, and elbow flexion. The most Dolutegravir order commonly reported test of lower extremity strength was the 1RM for leg press, estimated in three studies and measured in five studies (see Table 4 in the eAddenda). The pooled mean for 1RM was 67.6 kg (95% CI 61.2 to 73.8) for women on treatment and 95.8 kg (95% CI 88.3 to 103.4) for women off treatment (Figure 9). Heterogeneity

was found to be substantial for women off treatment only (I2 = 69%). Reported normative values are reported in weight pushed per kg of body weight, but for a woman weighing 70 kg, values for women on treatment fall into the ‘below average’ category for women aged those 50 to 59, while values for women off treatment fall into the ‘above average’ category for women aged 50 to 59 ( Table 2). 11 A leg-press protocol was also used to measure maximum isometric contraction and muscle endurance. Other protocols requiring resistance-training equipment include knee flexion and knee extension machines. Chair stands were also used as a functional measure of lower extremity function (n = 7), although pooled analysis was not possible due to the heterogeneity of protocols used. The TUG test was used to evaluate functional mobility in two included studies (see Table 5 in the eAddenda). However, the results from the two are not directly comparable as they used two different protocols: one used an 8-foot course and the other a 3-metre course.

The collected samples were stored at 4 °C. Starch degrading microbes were isolated using Strach Agar Medium (SAM). The isolates showing maximum clear halo zone were sub-cultured.7 Selective isolates with maximum starch degrading activities were identified up to species level.8 and 9 The most potent isolates were finally chosen for further studies. The inoculum for further enzyme modulation and other studies was prepared using Luria

Broth (LB) medium. The fresh overnight culture was used as an inoculum for the production of amylase.10 The inoculated medium was incubated at 37 °C for 48 h by shake flask fermentation method at 200 rpm. The culture broth was then centrifuge at 8000 × g 10 min at 4 °C. The free cell supernatant Doxorubicin datasheet was used as an extracellular crude enzyme. 11 Total protein concentrations were determined by Bradford’s method using Bovine Serum Albumin (BSA) as the protein standard.12 α-Amylase activity was determined by measuring the formation of reducing sugars released during starch hydrolysis. The amount of liberated reducing sugar was determined by Dinitrosalicylic acid (DNS) method. Glucose was used to construct Anti-diabetic Compound Library order the standard curve.4 Five percent bacterial inoculum was added aseptically to 500 ml of sterile growth

medium and incubated at 37 °C at 150 rpm. Twenty ml of culture was taken periodically for 48 h at every 6 h intervals. The amylase activity was determined in the culture filtrate. The effect of pH on amylase activity was determined at different pH (6.5, 7, 7.5, 8, 8.5 and 9) and the effect of temperature on enzyme activity was determined using different temperature (26 °C, 29 °C, 32 °C, 35 °C, 38 °C and 41 °C).11

Different carbon and nitrogen sources (both at concentration of 10 g/L) were used in minimal medium, pH 7 and incubated at 32 °C for 24 h. Similarly different amino acids like glycine, alanine, aspartic acid and cysteine were used in the medium for optimization.13 The culture filtrates were assayed for total protein content old and amylase activity. The culture filtrate was precipitated using 80% w/v Ammonium sulfate precipitation method.14 Then the precipitate was separated by centrifugation at around 6700 × g for 10 min. The pretreatment of the dialysis membrane was done Ashwini et al, 2011. Genomic DNA was extracted using phenol–chloroform extraction method. The PCR parameters for the amplification of 16S ribosomal DNA were optimized. 50 μl of PCR master mix contained universal primer set 27 F- (5′-AG AGT TTG ATC MTG GCT CAG-3′)/1492 R- (5′-G GYT ACC TTG TTA CGA CTT-3′), 10 mM dNTPS, 10× PCR Buffer, 1 U Taq DNA polymerase, 2 mM Mg+ and (100–200 ng) template DNA. PCR steps included initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 56 °C for 2 min, elongation at 72 °C for 1 min and final extension at 72 °C for 10 min. Approximately 1.5 kb amplicons were generated.