Using rpoB DPRA, we differentiated Mycobacterium tuberculosis com

Using rpoB DPRA, we differentiated Mycobacterium tuberculosis complexes

(MTC) from NTM with 235 base pair (bp) and 136 bp PCR amplicons in AFB smear-positive BACTEC cultures. The 136 bp rpoB duplex PCR amplicon was further digested with MspI and HaeIII (rpoB DPRA) to divide the NTM species into eight easily distinguishable groups (A–H) as described by Kim et al. [10]. Using two phenotypic characters (growth rate and photoreactivity on pigment production) and two simple biochemical assays (nitrate reduction test and Tween 80 hydrolysis test) [11], the mycobacterial species were identified. However, the sub-culture and biochemical tests for this algorithm took three weeks. In the present study, we developed RepSox purchase a rapid and effective algorithm for identification of mycobacteria by combined rpoB DPRA and hsp65 PRA with Alpelisib ic50 CE. Results Mycobacteria identification There were 376 AFB smear-positive BACTEC culture tubes (positive BACTEC cultures), including 200 MTC and 176 NTM-containing BACTEC cultures. A further 20 bacteria were MGIT positive but AFB culture smear

negative, and these were classed as contaminated and excluded from subsequent evaluation. By rpoB duplex PCR, all of the 200 MTC-containing BACTEC cultures and the 176 NTM-containing BACTEC cultures showed 235-bp and 136-bp PCR amplicons specific for MTC and NTM, respectively. The species were identified according to the flow chart shown in Figure 1. Figure 1 An flow chart of the identification of Mcobacterial species from clinical specimens by combined rpo B duplex PCR(DCR) and hsp 65 PCR-Restriction Fragment Length Polymorpgism analysis(PRA). Concordant results from rpoB DPRA and hsp65 PRA Combining rpoB DPRA and hsp65 PRA with computer-aided CE gave an accuracy rate of 100% (200/200) for MTC and 91.4% (161/176) for

NTM (Table 1). Table 1 Comparison of hsp65 RFLP, rpo B RFLP ADAM7 patterns, 16 S rDNA sequences and conventional biochemical identification in 361 isolates with concordant results rpoB RFLP pattern hsp65 RFLP pattern 16 S rDNA sequence identification Conventional biochemical BACTEC culture number Concordance rate       identification     T M. tuberculosis type 1 M. tuberculosis M. tuberculosis 200 100%(200/200)   NTM NTM NTM 161 91.4%(161/176) A M. abscessus type1 M. abscessus M. abscessus 29   A M. abscessus type 2 M. abscessus M. abscessus 41   A M. fortuitum type 1 M. fortuitum M. fortuitum 33   A M. fortuitum type 2 M. fortuitum M. fortuitum 2   A M. check details peregrinum type 1 M. peregrinum M. fortuitum* 5   A M. peregrinum type 2 M. peregrinum M. fortuitum* 8   A M. peregrinum type 3 M. peregrinum M. fortuitum* 1   A M. chelonae type 1 M. chelonae M. chelonae 1   A M. mucogenicum type 1 M. mucogenicum M. mucogenicum 2   A M. smegmatis type 1 M. smegmatis M. smegmatis 2   B M.

The sections represent regions of biofilm containing structured n

The sections represent regions of biofilm containing structured networks of fibers and sheets, but few bacteria. (A) The walls consisted of thin laminar structures (arrowhead) with globular material (arrow) accumulating in branching regions; CFTRinh-172 molecular weight scale bar = 500 nm. (B) In other regions of the biofilm, the wall-like structures had different thicknesses. The thin walls (arrowhead) were attached to SC79 purchase thicker walls (arrow); scale bar = 500 nm. (C) Different wall morphologies consisted of thin, straight walls (arrowhead) branching from thicker walled structures (arrows); scale bar = 500 nm. (D) The thicker walls were composed of globular amorphous masses (arrows) covered in part

by a distinct coating (arrowheads); scale bar = 200 nm. (E) and (F) The different components of the thicker walls consisted of globular masses (arrows) separated by and covered with thin coatings (arrowheads); scale bar = 500 nm. Biofilms are chemically heterogeneous Hydrated biofilms from multiple cultures were combined taking care to minimize the inclusion of spent media without disturbing the fragile structures. No further handling of the biofilms was carried out prior to freeze-drying in order to preserve the chemical integrity of the structures. Physical or chemical treatments of the samples learn more such as centrifugation, filtration, extraction, and ion exchange chromatography have the potential to significantly alter the biofilm

composition, thus biasing the results of the chemical analysis. The method described here is simple, convenient, minimally invasive, and is designed to provide representative samples for compositional analysis. Hydrated biofilms (0.9189 g) afforded 15.6 mg of dry material (16.0 17-DMAG (Alvespimycin) HCl mg g-1) consisting of biofilm and spent media, where-as spent media free of biofilm (1.9255 g) afforded 10.8 mg of dry material (5.6 mg g-1). Assuming that the dry material makes up a negligible proportion (1.7% in the case of biofilm plus media) of the mass of the hydrated sample, the media contribution to the mixed sample was estimated as 5.2 mg (0.9189

× 5.6), or 33% [(5.2/15.6) × 100%]. Background contributions from spent media to the chemical sample make-up were subtracted from the mixed biofilm-media samples according to eq. 1. This simple relationship was employed throughout to estimate biofilm composition. Results of the biofilm chemical analyses are summarized in Table 1. Table 1 Biofilm chemical composition. Analyte Analysis method Mass concentration (μg mg-1)a Calcium ICP-AES 29.9 Magnesium ICP-AES 10.1 Total proteins UV absorption 490 Total proteinsb Folin reaction (Lowry assay) 240 Acidic polysaccharidesc Phenol-sulfuric acid reaction 79 Neutral polysaccharidesc Phenol-sulfuric acid reaction 67 Nucleic acids UV absorption 46 DNA DAPI-fluorescence 5.4 aDry material. bMeasured as BSA. cMeasured as dextrose monohydrate. The principal IR absorption bands of the mixed biofilm/media sample are presented elsewhere [see Additional file 1].

PubMedCrossRef 26 Pearson WR, Lipman DJ: Improved tools for biol

PubMedCrossRef 26. Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 1988, 85:2444–2448.PubMedCentralPubMedCrossRef Epoxomicin purchase 27. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD: The Pfam protein families database. Nucleic Acids Res 2012, Database Issue 40:D290-D301.PubMedCentralPubMedCrossRef 28. Neumann L, Spinozzi F, Sinibaldi R, Rustichelli F, Pötter M, Steinbüchel A: Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules. J Bacteriol 2008, 190:2911–2919.PubMedCentralPubMedCrossRef

29. Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012, 9:671–675.PubMedCrossRef 30. Regensburger B, Hennecke H: RNA polymerase from Rhizobium japonicum . Arch Microbiol 1983, 135:103–109.PubMedCrossRef 31. Vincent JM: A Manual for the Practical Study of Root-Nodule Bacteria. Oxford, England: Blackwell Science Publications; 1970. [International Biological Programme Handbook No. 15] Competing interests The authors declare that they have no competing interests. Authors’ contributions Conception and design of the study: KY. Acquisition of data: YT and TS.

Analysis and interpretation of data: KT. Drafting the article: KY. Revising it critically for important intellectual BLZ945 nmr content: KT and ST. Final approval of the version to be submitted: All the co-authors. All authors read and approved the final manuscript.”
see more Background Mycobacterium

tuberculosis remains a threat to global RVX-208 health despite efforts directed towards its eradication. Although several works have been done in recent years towards understanding the genetic repertoire of this organism, many of its strategies involved in virulence, pathogenesis and resistance to both host pressure and antibiotics remain elusive [1]. Mycobacterial genome has been completely sequenced for over a decade [2]. However, the functions of many of its genes are annotated based only on similarity to known proteins using automatic annotation systems. This method of function annotation can be erroneous [3, 4]. Errors in automatic function annotation to genes in bacterial genomes are well documented. They often lead to misinformation that may hamper the understanding of the roles played by many bacterial genes [5–8]. Experimental characterization of additional mycobacterial proteins is needed to aid deeper understanding of the organism. Histidine phosphatase superfamily is a large family of proteins with diverse functions that are important. This superfamily comprises two branches. The larger branch consists of proteins which function in metabolic regulations, intermediary metabolism and developmental processes.

0 and were applied to a Q-Sepharose column The proteins were elu

0 and were applied to a Q-Sepharose column. The proteins were eluted with 15 column volumes of buffer containing 0.1% DDM, 10 mM Tris-HCl at pH 7.0, and an increasing concentration of NaCl (linear selleck chemicals llc gradient of 0-300 mM; Additional file 1). The peak fractions were applied to a hydroxyapatite column for separation. The proteins were eluted with 3 column volumes of buffer containing 0.1% DDM and an increasing concentration of NaPi at pH7.0 (stepwise gradient of 20, 50, 100, 150, 200, 300, and 400 mM; Additional file 2). Enzyme activities Cytochrome oxidase activity was assayed at 60°C by measuring oxidation of a yeast cytochrome c (Sigma-Aldrich, St. Louis MO), which had been reduced with sodium dithionite,

in a final volume 800 μL containing a suitable amount of enzyme, 20

mM NaPi at pH 7.0, and 10 μM yeast cytochrome c. The oxidation of reduced cytochrome c was followed by measuring the decrease in absorbance at 549 nm, and activity was calculated using a millimolar absorption coefficient of 21.2 mM-1 cm-1 [24]. N, N, N ‘, N ‘-Tetramethyl- p -phenylenediamine (TMPD) oxidase activity was assayed by measuring the increase in absorbance at 562 nm using a mixture of 25 mM TMPD, 0.1 M NaCl, and 50 mM NaPi at pH 6.5, and calculated using a millimolar absorption coefficient of 10.5 mM-1 check details cm-1. To avoid the auto-oxidation of TMPD, the assay was CB-5083 performed at 40°C. Menaquinol oxidase activity was assayed at 40°C by measuring the oxidation rate of menaquinol-1, which had been reduced with sodium dithionite, in a final volume of 700 μL containing a suitable amount of enzyme, 20 mM NaPi

at pH 7.0, 0.1% (w/v) DDM, 1 mM EDTA, and 0.2 mM menaquinol-1. The oxidation of reduced menaquinone was followed by measuring the increase in absorbance at 270.7 nm, and the activity was calculated using a millimolar absorption coefficient of 8.13 mM-1 cm-1. Electrophoretic analyses Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was performed according to the method of Schägger et al. [25]. Nondenaturating electrophoresis was started at 100 V until the sample was within the stacking gel and continued with the voltage and current limited to 350 V and 15 mA, respectively. For two-dimensional Farnesyltransferase analysis, a slice of the BN-PAGE gel was excised and soaked in 1% sodium dodecyl sulfate (SDS) and 1% mercaptoethanol buffer for 1 h and then embedded in a separating gel containing 15% acrylamide. Two-dimensional analysis was performed at room temperature with the current limited to 20 mA. SDS-PAGE was performed according to the method of Laemmli [26]. The gel was stained for protein with CBB and for heme with o -toluidine in the presence of H2O2. Gels were immersed in a solution containing 1% (w/v) o-tolidine, 80% (v/v) CH3OH and 10% (v/v) CH3COOH for 10 min, and then H2O2 was added at final concentration of 1% (v/v).

16 Upper end 823 0x Shaft 823 2x Unspecified 823 8x 6 Wrist (clo

16 Upper end 823.0x Shaft 823.2x Unspecified 823.8x 6. Wrist (closed) Pathologic 733.12 Forearm upper end 813.0x Shaft 813.2x Lower end 813.4x Unspecified 813.8x 7. Spine/vertebral (closed) Pathologic 733.13 Cervical, closed 805.0x Dorsal, closed 805.2x RG7112 ic50 Lumbar, closed 805.4x Unspecified, closed 805.8x Statistical analysis Patients were stratified into two groups, FRAC and ICD-9-BMD, based on reason for inclusion. Descriptive statistics,

including proportion of patients treated, were used to characterize the baseline demographic and clinical characteristics of patients in both groups. A logistic regression was used to identify predictors of osteoporosis treatment with an oral bisphosphonate (risedronate, alendronate, or ibandronate). Patients were identified as treated if they had a prescription for one of the

three drugs on the index date or up to 90 days post-index date. Regressions were run Cytoskeletal Signaling inhibitor separately for each of the two patient groups. Independent variables included age at index date (50–64, 65–74, and 75+), BMI (≤24 kg/m2, 25–29 kg/m2, 30–34 kg/m2, and 35+ kg/m2), smoking status, excessive alcohol consumption, fall history, insurance status (Medicare, private insurance, or no insurance), presence of an order for a BMD test, and BMD find more T-score. The value for the BMD T-score variable was the test result for the hip, if available. If the hip T-score was not available, a spine test result was used, and if neither a hip or spine result was available, a forearm score was used. Values for the BMD T-score variable included test results within the first 90 days after the index date and was dichotomized based on

whether the value was greater than or less than or equal to −2.5. Therefore, Bay 11-7085 patients in the FRAC group, who by definition did not have a T-score ≤−2.5 on the index date, may still have a value for this variable below this threshold if it was measured in the first 90 days post-index. Furthermore, while it was not possible to link the cause of the fracture for patients in the FRAC group to a specific fall, if the fracture was the result of a fall, that fall would be captured by the fall history variable. Also included were diagnoses of comorbidities associated with bone health such as aortic atherosclerosis, diabetes, thyroid disease, and malnutrition. Indicators for the use of drugs over the study period whose exposures are associated with fracture risk were also included (e.g., chemotherapy, oral corticosteroids, thyroid replacement therapy, and furosemide therapy). Finally, a Charlson Comorbidity Index (CCI) score was calculated for each patient based on comorbidities documented on or one year prior to their index date [26]. Initially, a forward selection process was undertaken by running univariate regressions with each independent variable. Variables whose coefficients had p values of ≤0.10 were chosen to be included in the full multivariate regression.

RPMI 1640 medium

RPMI 1640 medium Capmatinib cell line containing 10% FBS was replaced by serum-free Opti-MEM (GIBCO, Invitrogen, USA) at 8 h later. HiPerFect Transfection Reagent and Negative control siRNA were purchased from Qiagen Technology Co. Ltd (Shanghai, China). Transfection compounds were prepared in three groups as follows: siRNA group (100 μl Opti-MEM, 6 μl HiPerFect Transfection Reagent and 5 μl JMJD2A siRNA), negative control group (100 μl Opti-MEM, 6 μl HiPerFect Transfection Reagent and 5 μl negative control siRNA) and blank control group (100 μl Opti-MEM). Transfection compounds were placed at room temperature for 10 minutes and then dropped onto 6-well plates. Bulk volume of the compounds

was 2200 μl per well. Both Opti-MEM and transfection compounds were replaced by complete medium at 24 h after transfection. FAM-siRNA was transfected to measure the efficiency of transfection simultaneously GDC-0941 purchase according to the manufacturer’s instructions. Quantitative real-time PCR Total RNA of three groups was extracted respectively with the RNAiso Reagent kit (TaKaRa, Dalian, China) at 48 h after transfection. cDNA was I-BET-762 in vitro generated by reverse transcription of 2 μg of total RNA using random primers and PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Dalian, China) in a total reaction volume of 40 μl according to the manufacturer’s instructions. The sequences of forward and reverse oligonucleotide primers, specific to JMJD2A and housekeeping genes, were designed

using Primer5 software. The primers Glutamate dehydrogenase used are: 5′-TGTGCTGTGCTCCTGTAG -3′ and 5′-GTCTCCTTCCTCTCCATCC -3′ for JMJD2A; 5′-TGACGCTGGGGCTGGCATTG -3′ and 5′-GCTCTTGCTGGGGCTGGTGG -3′ for GAPDH. Primers were synthesised by Shanghai Daweike Biotechnology Co. Ltd (Shanghai, China). Real-time quantitative PCR was performed in an ABI PRISM 7500 Real-Time System. A 10-fold dilution of each cDNA was amplified in a 20-μl volume, using the SYBR Premix Ex TaqTM Perfect Real Time (TaKaRa, Dalian, China), with 0.2 μM final concentrations of each primer.

PCR cycle conditions were 95°C for 30 s, and 40 cycles of 95°C for 5 s and 60°C for 34 s. The amplification specificity was evaluated with melting curve analysis. Threshold cycle Ct, which correlates inversely with the target mRNA levels, was calculated using the second derivative maximum algorithm provided by the iCycler software. For JMJD2A, the mRNA levels were normalized to GAPDH mRNA levels [9]. Western blot At 72 h after transfection, cells in different treatment groups were homogenized in Western blot analysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM PMSF, 0.28 kU/L aprotinin, 50 mg/L leupeptin, 1 mM benzamidine and 7 mg/L pepstain A. The homogenate was then centrifuged at 12, 000 rpm for 10 min at 4°C and the supernatant was retained and preserved at -80°C for later use. Protein concentration was determined using a BCA kit (Pierce).

In NSCLC, chemotherapeutic treatment can damage DNA through vario

In NSCLC, chemotherapeutic treatment can damage DNA through various mechanisms, the lack of AP24534 mw functional BRCA1 can lead to increased

sensitivity of the tumor cells to molecular damage, demonstrating that BRCA1 represents a predictive marker of chemotherapy response in NSCLC [6]. Ribonucleotide reductase subunit M1 (RRM1) is located on chromosome segment 11p15.5, it is a region with a frequent loss of heterozygosity in NSCLC. It is a component of ribonucleotide reductase, which is required for deoxynucleotide production and is also the predominant cellular determinant of the efficacy of gemcitabine, which make it to be the molecular target of gemcitabine [7, 8]. Along with the use of antitubulin agents such CP673451 cost as taxanes and vinorelbine, study shows there are a number of tubulin isotypes in humans, and found that class III β-tubulin (TUBB3) among them is expressed in a proportion and related to clinical outcome [9]. The expression of selleck chemicals llc TUBB3 is associated with resistance of paclitaxel and docetaxel, no matter in vitro or in clinical research [10, 11]. Changes

of gene mRNA expression during carcinogenesis may lead impact of the diagnosis, treatment, and prevention of NSCLC, it is important to understand these changes. So, in this study, we use RT-PCR to examine the expression of ERCC1, BAG-1, BRCA1, RRM1 and TUBB3 in tumor samples from patients with resected NSCLC not receiving adjuvant chemotherapy. We analyzed the relationships of these genes expression in tumors about survival time and response to chemotherapy to determine whether the expression of these molecules could be used as prognostic factors of progression-free and overall survival in this cohort of

patients. Methods Patient data A total of 85 patients who underwent curative surgery for NSCLC between August 2007 and April 2009 were enrolled into this study, including 85 tumor tissues and 34 adjacent tissues respectively. Among them there were 60 males and 25 females, aged 24-84 (mean 57) years. According LY294002 to WHO Classification (2000), there were 25 squamous, 60 adenomatous, with 58 moderate and well differentiated (G1-G2) and 27 low differentiated (G3). Because there were only 4 cases of stage IV patients who all had surgery after single brain metastasis resected firstly, and there were no patients of stage IIIb. On account of stage IV patients were too few, so we combined 48 cases as staged I-II and 37 III-IV based on the revised AJC staging for lung cancer (1997). 28 cases had intra-thoracic lymph node metastasis (N1-N2), and 57 were negative lymph node metastasis. Additional information of surgery and chemotherapy status were all showed in (Table 1). The paracancerous tissues (defined as more than 5 cm away from the carcinoma tissue) taken from 34 cases were used as controls.

[12] in a mice model However, these anti-inflamatory effects see

[12] in a mice model. However, these anti-inflamatory effects seen in vivo are not as powerful as those previously described in vitro [13]. The differences are even greater when the in vivo data is obtained from athletes [14–16]. Quercetin supplementation improves running time to fatigue by stimulating mitochondrial biogenesis in mice [6]. However, this effect has not been observed in humans [16–18]. Research has shown improvements of 3.9% in VO2 peak and 13.2% in time to fatigue [19], as well as 2.9% in a selleck compound maximal 12-minute test after an hour of preload [18] in untrained

subjects. These findings are in contrast to those of previous studies [11, 17, 20]. When athletes are studied, most research has failed to find an ergogenic effect [15, 16], in contrast to that of a study of elite cyclists, who exhibited Vactosertib order an improvement of their aerobic performance [21]. Finally, effects of quercetin on pre-exercise and post-exercise blood lactate have not been reported [22]. Based on the data provided, the question arises: could quercetin be an ergogenic supplement for athletes or untrained subjects? LDK378 Our primary goal is to study, for the first time and using a rat model, the effects of both endurance training and chronic quercetin supplementation on 1) endurance capacity, VO2 peak, and lactate production, 2) endurance

training progress, and 3) distance covered in a low-intensity treadmill test and in a high-intensity treadmill test. Methods Animals and experimental design Thirty-three young (three week old) male Wistar rats were randomly allocated into four groups: quercetin and endurance training (QT, n=9), placebo and endurance training (PT, n=8), quercetin and sedentary (QS, n=8), and placebo and sedentary (PS, n=8). Animals, with an initial body weight of 150 (SD=10) g, were housed in individual stainless steel metabolism cages. The cages were located in a well-ventilated thermostatically

controlled room Oxymatrine (21 ± 2°C), with relative humidity ranging from 40 to 60%. A reverse 12 h light-12 h dark cycle (08.00-20.00 hours) was implemented to allow exercise training during the day. Throughout the experimental period, all rats consumed water and food ad libitum. Two weeks before the experimental period, rats were allowed to adapt to the diet and experimental conditions, and a week before the experimental period, rats had three days of acclimation to the treadmill. Body weight was measured twice per week during this time. After six weeks of treatment we performed two different exercise tests. Tests were carried out after the treatment so that we could compare four different conditions without assessing the effect of training. The reason for choosing a rat model is that a previous study showed that sedentary mice exhibited higher endurance performance with quercetin intake than with placebo [6].

The amplified products were electrophoresed

The amplified products were electrophoresed learn more on a 1.25% agarose gel (Invitrogen, USA). DNA extracts of G. duodenalis from an axenic

culture was used as positive control throughout the study. 5. DNA cloning and sequencing The PCR products were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA) according to the manufacturer’s instruction and directly sequenced. Both strands of the entire fragments were sequenced with primers GDHeF and GDHiR, then manually assembled in BioEdit version 7.0.1. When the one singleton substitution was found, the sequencing was repeated with the PCR product from the independent PCR amplification. If a superimposed signal in chromatograms was detected, showing incorporation of the two bases resulting from co-amplification, cloning of this PCR product was performed to confirm the existence of the multiple templates. To clone, the purified PCR product was ligated into pGEM-T Easy vector (Promega, Madison, USA). Ligated product was introduced into JM109 competent cells by

transformation. The recombinant plasmids were purified from 10 positive clones of each sample using the HiYield Plasmid mini kit (RBC Bioscience, Taiwan) mTOR inhibitor and sequenced using universal primer SP6. DNA sequencing was conducted by 1st Base Pte. Ltd., Singapore. The novel nucleotide substitutions obtained from clones corresponded to alleles if the substitution at that position occurred two or more times. 6. Sequence analysis On all analyses, the priming sites were trimmed from both ends of all sequences which reduced the fragment size to 414 bp. All sequences were multiple aligned with the default option using CLUSTAL X, version 2.0.12 [22] and analyzed separately based on their assemblages, assemblage A and assemblage B. Each assemblage was both analyzed separately depending on the origins of the isolate and together. The partial sequences Tolmetin of the gdh gene of the G. duodenalis ATCC 50803 assemblage A isolate WB and G. duodenalis ATCC 50581 assemblage B isolate GS, acquired from GiardiaDB: The Giardia Genomics Resource http://​Acadesine giardiadb.​org/​giardiadb/​,

were used as reference sequences. The subassemblages were assigned through Bayesian inference constructed using MrBAYES Version 3.1.2 [23]. The reference sequences of assemblage AI (accession no. L40509), AII (accession no. L40510), BIII (accession no. AF069059), and BIV (accession no. L40508) were also implemented in the tree. The analysis of synonymous and non-synonymous amino acid substitutions was performed using MEGA version 4 [24]. The level of nucleotide divergence (K), including synonymous (Ks) and nonsynonymous (Ka) divergence rates, and number of allele were calculated using DnaSP version 5 [25]. This program was also used to quantify the level of genetic variation among Giardia isolates collected from different regions by the Wright’s fixation index (F ST).


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