All authors read and approved the final manuscript.”
“Background Campylobacteriosis is one of the most important foodborne diseases worldwide. The number of reported cases varies by country. For instance, New Zealand had FG-4592 in vivo the highest incidence with 161.1 cases for every 100,000 population in 2008 [1]. Canada had an incidence of 36.1 cases for every 100,000 person per years [2], and European countries have an overall incidence of 48 cases per 100,000 population [3]. In Scotland, there were 95.3 reported cases per 100,000

in 2006 [4]. In the US, campylobacteriosis is the third most important bacterial foodborne disease, with an incidence

of 12 cases per 100,000 [5]. Campylobacter spp. are still found at high prevalence in Vorinostat order retail broiler carcasses [6, 7] and in retail broiler meat [8–10]. In the USA, the U. S. Department of Agriculture Food Safety and Inspection Services (USDA FSIS) has recently updated the compliance guideline for poultry slaughter to make the regulations related to Salmonella detection more selleck inhibitor stringent and to enforce the implementation of a performance standard for Campylobacter spp. [11]. Although there have been recent

reports reviewing the incidence of campylobacteriosis per year [5] and the prevalence of Campylobacter spp. in processed carcasses [7], there are no recent reports on the prevalence of these bacteria in retail broiler meat. In addition, the reports of prevalence are always presented without analyzing the data by nominal variables, i.e. processing plant, product, season, state and store that may influence the prevalence of these bacteria in retail broiler meat. This publication summarizes the prevalence of Campylobacter Janus kinase (JAK) spp. in skinless, boneless retail broiler meat from 2005 to 2011. Besides describing the prevalence per year, the prevalence by brand, plant, product, season, state, store, and Campylobacter spp. found in the products are described. In addition, the results of typing these Campylobacter isolates by pulsed field gel electrophoresis (PFGE) to determine the DNA relatedness of isolates from the same processing plants but from different years are presented.

All of the information concerning the trajectory of these times was collected every 5 ps. The equilibration of the trajectory was checked by monitoring the equilibration of the quantities, such as the RMSD of non-hydrogen atoms with respect to the initial structure. Fludarabine solubility dmso Analysis of the total energy, potential energy and kinetic energy were all obtained using GROMACS software. RMSD values between final and LY3039478 nmr template structures also helped to identify the common segments, which corresponds to the structurally conserved region. The average structure of the entire trajectory was also determined using the g_rms algorithm [68]. The first 10 ns of the trajectory

were not used to determine the average structures. All of the water molecules were removed from the selected structures to proceed with the docking simulations in the next step. Molecular docking By using the structures of PbMLS-interacting proteins determined by MD as described above, a global search find more of protein-protein interactions was performed using GRAMM-X software [69]. The Protein-Protein Docking Web Server v.1.2.0 was used to perform rigid docking. Simulations were performed with no pre-conceived bias toward specific residue interactions, and the best model-structure of each complex (PbMLS + PbMLS-interacting proteins) was selected. Refinement of MD MD simulations of the complexes were performed to improve the

orientation of their side chains and to minimize the high-magnitude repulsive interactions between atoms. Short simulations were performed for the complexes defined by the GRAMM-X software, again using GROMACS software, with the same force field and solvent model Reverse transcriptase previously used to define the 3D-structures of each protein. The system was defined by a cubic box with periodic boundary conditions, and a 9 Å cut-off for non-bond interactions was used for electrostatic interactions treated by the Particle Mesh Ewald method. Overlapping water molecules were deleted, and the systems were neutralized by adding counter ions. Initially, the

system was subjected to minimization using steepest descent energy. The simulations were completed when the tolerance of 1000 kJ/mol was no longer exceeded. After minimization, the system was subjected to a 100 ps simulation in the NVT ensemble and then was immediately subjected to a 100 ps simulation in the NPT ensemble. For both stages, T = 300 K, and the thermostat relaxation constant = 0.1 ps; additionally, a Berendsen thermostat, 1 atm pressure, a time-step of 2 fs and position restraint of the complex were used. After that step, the system was subjected to an MD run in the NPT ensemble. The simulations were performed for 1 ns with a constant temperature of 300 K, 1 atm pressure, a time-step of 2 fs and without any restriction on the complex conformations.

However, some genes that were downregulated in 13124R were upregulated in NCTRR. qRT-PCR analysis confirmed that the transcription of these genes, which included toxin genes for phospholipase C (PLC),

perfringolysin O (PFO), collagenase and clostripain, were affected differently in the two mutants. Similarly, the production of these enzymes and the toxicity Selleck Captisol of the culture supernatants decreased in 13124R and increased in NCTRR. It appears that gatifloxacin resistance selection resulted in alteration of global gene transcription in C. perfringens and that the effect was strain-specific. The changes in the levels of global gene expression due to the response to fluoroquinolone exposure may be governed by complex regulatory processes. Both resistant strains harbored some common and some unique mutations in fluoroquinolone target genes. These enzymes are involved in the DNA supercoiling process that plays an essential role during gene transcription [38, 39]. Although neither of the resistant strains was a clinical isolate, some of the mutations found in the resistant strains were the same as those found in fluoroquinolone-resistant mutants of E. coli obtained from clinical samples, which were also the same as those

found in fluoroquinolone-resistant mutants of E. coli generated in the laboratory [29, 40]. The expression of a number of genes is affected by supercoiling [19] and aberrant expression of these genes occurs when DNA supercoiling

Metalloexopeptidase has been altered by gyrase mutation(s). Alleles of gyrA that reduce DNA supercoiling have been shown to generate metabolic defects and reduce fitness of gyrase mutant strains [38, 41]. Furthermore, Doramapimod cell line because fluoroquinolones are DNA-damaging agents, in addition to inducing mutation in target genes, changing DNA superhelicity, they may also induce the expression of DNA repair genes via the SOS response, which may lead to phenotypic changes [15, 17–20]. Cirz et al. [15] characterized the global transcription response of S. aureus to ciprofloxacin and, among other changes, found induction of the SOS response, upregulation of the TCA cycle and downregulation of α-hemolysin and a leukocidin family toxin. The positive regulators of transcriptional responses for stress and toxin genes were also downregulated [15]. In C. perfringens, although the expression of several this website virulence genes decreased in one resistant mutant (13124R), it increased in another (NCTRR). The transcription of various genes, including toxin genes, is regulated by virR and virS[32, 42, 43]. VirS is a sensor histidine kinase, which autophosphorylates in response to extracellular signals, and VirR is a response regulator [32, 42, 43]. These two genes, along with vrr (which is an RNA regulator virR-RNA), are implicated in controlling gene transcription [44] and were upregulated in NCTRR. In 13124R, transcription of VirR did not change, and virS and vrr were downregulated.

This shared morphology might represent an adaptation to growing near active resin flows: the perennial ascocarps can effectively rejuvenate in situations where they happen to be partly submerged in fresh exudate. All three species commonly live on cankers and wounds which exude resin over extended periods. It seems unlikely that the ascomata of resinicolous Chaenothecopsis species could rejuvenate after being rapidly and completely submerged

in fresh sticky resin. Even the fossil specimens had first produced fruiting bodies on hardened resin and then GSK2118436 manufacturer had subsequently been covered by a thick layer of fresh exudate. This raises the question of what then triggers the proliferation in partly submerged ascocarps and those ascocarps only growing close to fresh resin. It has been shown that some fungi react to the volatile compounds produced by other fungi when competing for resources (Evans et al. 2008). It is also known that fresh resin contains high levels of volatile compounds, mainly monoterpenes and sesquiterpenes, when compared to older, semisolid exudate, and that the hardening of resin is directly related to the loss of such compounds (e.g. ACP-196 molecular weight Langenheim 2003; Ragazzi and Schmidt 2011). An ability to detect and respond to the presence of volatile resin compounds in the environment would give the Chaenothecopsis

species time to prepare for a potential burial in freshly exuding resin. It seems feasible that some resinicolous fungi could begin to branch when the concentration of volatile resin compounds in their typically sheltered microenvironment Selleckchem 4SC-202 is sufficiently high as to indicate that a fresh resin flow may be imminent. In other fungi the differentiation of fruiting bodies is commonly triggered by the perception of some change in environmental conditions, such as light, pH, Cyclic nucleotide phosphodiesterase oxygen etc. (Busch and Braus 2007). The hyphae of extant resinicolous fungi commonly penetrate and grow into semisolid resin. Evidence

of inward growth of fungal hyphae is also preserved in numerous worldwide amber fossils since the Paleocene (personal observation), but no evidence of a similar capability has yet been found prior to the Cretaceous-Paleogene boundary. Cretaceous amber pieces from several different deposits may contain abundant filaments that grew from the resin surface into liquid resin, but all of these have been identified as filamentous prokaryotes (see Schmidt and Schäfer 2005; Schmidt et al. 2006; Girard et al. 2009a, b; Beimforde and Schmidt 2011), not as fungal hyphae. This suggests that this special niche was either occupied by prokaryotes in the Mesozoic or that Chaenothecopsis species (if already existent) and other ecologically similar fungi did not yet exploit resin substrates. Conclusions Fossil evidence of inward growth of fungal hyphae into plant exudates has not been identified from Mesozoic ambers, suggesting a relatively late occupation of such substrates by ascomycetes.

LS and GF carried out experimental work on adhesion factors. EG and AN performed the initial isolation of A. baumannii. LP supervised the genetic characterization of the isolates. PL supervised the experiments related to the identification of the adhesion factors and wrote the manuscript, which was revised and approved by all authors.”
“Background Enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic and enteroaggregative Escherichia

coli are categories of enteric E. coli that have been unequivocally Evofosfamide in vitro associated with diarrhoeal disease through human challenge studies and/or outbreak investigations [1]. Regarding other potentially diarrhoeagenic categories of E. coli, the most evidence for enterovirulence has been compiled for diffusely adherent E. coli (DAEC). However, the basis for DAEC pathogenicity is not well understood. The category is heterogeneous and although some studies have shown an association of DAEC with diarrhoea, Staurosporine research buy others have not [2]. Two DAEC strains did not elicit diarrhoea upon human volunteer challenge and no outbreaks of DAEC-associated illness have been documented to date [3]. Enteroaggregative E. coli (EAEC) is another heterogeneous diarrhoeagenic E. coli category. Convincing

epidemiological information from EAEC outbreaks exists, and at least one strain was diarrhoeagenic in some human volunteers, however the category is very diverse (reviewed in references [4] and [5]). Compared to other diarrhoeagenic E. coli categories, EAEC and DAEC pathotypes were both described relatively recently and their epidemiology, risk factors and pathogenesis are still in early stages of investigation. Few epidemiological studies seek these categories because the Gold Standard test for their detection, the HEp-2 adherence assay, is cumbersome. This tissue culture-based

test requires expensive facilities and technical expertise that are not universally available. An improved understanding of the importance of diarrhoeagenic E. coli in human disease will depend upon BIBW2992 in vivo reliable epidemiological data and on channelling of strains identified into molecular Phosphatidylinositol diacylglycerol-lyase pathogenesis research. Accordingly, efforts have been made to develop more widely applicable methods to detect EAEC and DAEC. Baudry et al. tested fragments from the large plasmid of EAEC strain 17-2 and identified a 1 Kb fragment, CVD432, which was 89% sensitive and 99% specific for EAEC strains in their collection [6]. Subsequently, this probe has continued to show specificity for EAEC but its sensitivity has varied between 15 and 90% in different studies [4]. Bilge et al. [7] used a different approach to generate a diagnostic probe for DAEC. They identified, cloned and characterized the F1845 adhesin from DAEC strain C1845. The F1845 adhesin belongs to the Afa/Dr family and is encoded by a five-gene cluster [2]. Bilge et al.

Other investigators may have received portions of these tissue samples. Patient diagnostic and treatment information were made available for each tissue. Tissues were collected as snap frozen specimens stored at -80°C. Sample preparation and genomic DNA isolation Each snap frozen tissue was sectioned on a bed of dry ice to ensure minimal thawing during sample preparation. An approximately 30-50 mg piece of tissue was cut and an adjacent piece of tissue was removed for formalin fixation and paraffin embedding

for subsequent histological processing. Genomic DNA was isolated from tissue samples via homogenization in ice cold lysis buffer [10 mM Tris pH 8.0, 0.1 M ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS), 100 μg/mL Proteinase PD0332991 in vitro K, 25 μg/mL RNAase]. Subsequent phenol-chloroform extraction was carried out as previously described [24]. Integrity and concentration of each resulting DNA sample was assessed selleckchem by agarose gel electrophoresis. Sequencing primer design The known selleck chemicals llc coding region of SOSTDC1 is contained within two exons. Other potentially transcribed areas have been identified in the University of California Santa Clara Genome database [25–27]. Two of these potential exons occur upstream of the coding region and an additional exon occurs between the known coding exons for a total

of five putative exons or regulatory regions at this locus (see Additional file 1). Primers were designed for direct sequencing for a total of 13 pairs of direct sequencing primers (see Additional file Amoxicillin 2). All primers were synthesized by Integrated DNA Technologies (IDT). PCR amplification and direct sequencing Each direct sequencing primer pair was used to amplify all five putative regions of interest in each normal and tumor sample via PCR. PCR was performed in 40 μL reactions using 60 ng of genomic DNA, 15 pmol of both the forward and reverse primer, 4-5U of Taq polymerase (Life Technologies), 1.5 mM MgCl2, 200 μM dNTPs. Depending on prior reaction optimization, general cycling conditions were:

94°C 4 min, followed by 25-30 cycles at 94°C for 1 min, Tanneal for 1 min, and at 72°C for 1 min; and finishing with a single extension cycle at 72°C for 5 min. PCR products were purified using the Quickstep 96-well PCR purification kit (Edge Biosystems). DNA sequencing was performed using the ABI BigDye Terminator sequencing kit (Applied Biosystems, Inc.) Each 10 μL sequencing reaction contained 10-50 ng of purified PCR product, 1.5 pmoles of sequencing primer, 1 μL of BigDye Terminator mix, 1.5 μL of 5 × sequencing dilution buffer (400 mM Tris pH 9.0, 10 mM MgCl2) and water to volume. Cycling conditions were 94°C for 1 min; 25 cycles at 94°C for 30 sec, 50°C for 30 sec, and 60°C for 4 min; and finishing with a single 72°C extension step for 5 min. The sequencing reactions were run on an ABI 3730XL DNA sequencer and data were analyzed using Sequencher software (GeneCodes, Version 4.7).

Therefore,

the clear distinction of halocline ciliate communities from brine communities is not an unexpected result. However, it is surprising that the environmental variables we measured had a minor contribution to differences among the individual brine ciliate communities. In the CCA analyses (Figure 3) the different brine communities were spread out along the y-axis. This axis, however, does not represent an environmental gradient. This is surprising, considering that different types of salts may have different physiological effects [61] and therefore, should require different adaptation strategies in halophiles. Basically, we can assume Adriamycin molecular weight two scenarios: first, for isolated evolution as described in [62], the scenario starts with a seed taxon. After physical separation of the original habitat into two Selleckchem PI3K Inhibitor Library habitats neutral mutations are changing the seed taxon in these habitats independently. These neutral mutations are of minor nature considering the

time scale of the basins’ geological histories. From this event we would expect similar taxon groups with only minor genetic changes in both habitats. As mentioned above, each eighth taxon recorded in our study (Additional file 3: Table S1) falls into this category. In the second scenario (environmental filtering) we have the same ‘seed bank’ community for different basins. Through environmental filtering (different hydrochemistries of the basins) some taxa may go extinct, others have the genomic potential to adapt to some specific hydrochemistries, Mocetinostat datasheet while others are genomically equipped for adaptation Adenosine to other environmental conditions. In this case we would find taxa differing on higher taxonomic (genetic) hierarchies. This is the case for 34 of 102 detected taxon groups (Additional file 3: Table S1). We cannot rule out all environmental factors from causing differences between the ciliate communities because we did not measure all

possible environmental factors, but only the hydrogeochemical factors that account for the most pronounced and obvious differences. This suggests that (1) other hydrochemical variables we did not measure are leading to this separation, or (2) that biotic interactions may explain some of the differences between brine ciliate communities. Even though interactions of top-down and bottom-up factors in shaping community structures of aquatic microbes are still poorly understood [63] some well known biotic interactions could be considered. Such biotic interactions may be, for example, parasitic relationships between organisms like amoeboid parasitic forms that can shape the composition of cyanobacterial species in lakes (Rohrlack et al., unpublished data).

Figure 1 Time to exhaustion (individual responses,

A and mean values, B) after the ingestion of LGI, HGI and control meals (mean ± SEM). LGI: Low Glycemic Index; HGI: High Glycemic Index. RPE, heart rate and ventilation There was no significant main effect of trial or time by trial interaction for RPE (Figure 2A). selleck products However, there was a significant main effect of time (P < 0.001, η 2 = .98, observed power = 1.00). RPE levels increased significantly at 20 min and remained significantly elevated until exhaustion for all trials. There were no significant differences at rest between the three trials for heart rate (Control = 68.0 ± 2.6 bpm, LGI = 66.3 ± 4.2 bpm, HGI = 66.5 ± 3.4 bpm). There was no significant main effect of trial or time by trial interaction for heart rate (Figure 2B) and ventilation (Figure 2C). Crenolanib mouse However, there was a significant main effect of time for heart rate (P < 0.001, η 2 = .97, observed power = 1.00), and ventilation (P < 0.001, η 2 = .98, observed power = 1.00). Pairwise comparisons revealed significant differences between the 10 min and exhaustion time points for all trials for heart rate and ventilation. Figure 2 RPE, heart rate and ventilation responses during exercise after LY3023414 research buy the ingestion of LGI, HGI and control meal (mean ± SEM). LGI: Low Glycemic Index; HGI: High Glycemic Index.a Significantly different from 10 for the HGI group (P

< 0.05),b Significantly different from 10 for the LGI group (P < 0.05),c Significantly different from 10 for the control group (P < 0.05). Substrate oxidation There was no significant main effect of trial or time by trial interaction for respiratory quotient (RQ; Figure 3A). However, there was a significant main

effect of time (P < 0.001, η 2 = .97, observed power = 1.00). RQ appeared significantly elevated only at exhaustion with no significant difference between the three trials. Carbohydrate Gefitinib and fat oxidation rates (Figure 3B) was not different between the three trials during exercise. Figure 3 Respiratory quotient and substrate oxidation rate during exercise after the ingestion of LGI, HGI and control meal (mean ± SEM). LGI: Low Glycemic Index; HGI: High Glycemic Index.a Significantly different from 10 for the HGI group (P < 0.05),b Significantly different from 10 for the LGI group (P < 0.05),c Significantly different from 10 for the control group (P < 0.05). Lactate, glucose and insulin There was no significant main effect of trial or time by trial interaction for lactate (Figure 4A). However, there was a significant main effect of time (P < 0.001, η 2 = .92, observed power = 1.00). Lactate levels increased significantly at 20 min of exercise and remained significantly elevated until exhaustion for all trials. Figure 4 Lactate, glucose and insulin responses during exercise after the ingestion of LGI, HGI and control meal (mean ± SEM). LGI: Low Glycemic Index; HGI: High Glycemic Index.

7 mN on Si(100) surface and 2.0 mN on the other two crystal planes (indicated by arrows). Based on the Hertzian contact model [15], the corresponding maximum contact pressure (P 0) was estimated as 10.9 GPa for Si(100), 13.4 GPa for Si(110), and 14.2 GPa for Si(111), respectively. Since the hardness of Si(100), Si(110), and Si(111) was measured as 11.3, 13.0, and 13.2 GPa with the triboindenter, the calculated critical pressure is very close to the hardness of monocrystalline

silicon with different crystal planes [8, 16]. With the increase in F n, although Apoptosis inhibitor the value of P 0 attains to that of the hardness, the average pressure on the contact area may be still lower than that on the hardness. Hence, the scratch with both hillock and groove will be produced, and the hillock will become larger as the load increased. With the further increase in the load, groove formation will be dominant, and hillock will disappear because of the severe plastic

deformation. Therefore, when the contact pressure is less than the hardness of the monocrystalline silicon, the friction-induced hillock can be created on silicon surfaces with various crystal planes. Figure 1 Evolution of the scratches on (a) Si(100), (b) Si(110), and (c) Si(111) surfaces. Selleck TH-302 The scratches were produced at a linearly increasing load from 0.3 to 6.0 mN. Each AFM image (2 × 2 μm2) was taken from the appointed segment of the same scratch on silicon with a given crystal plane. The arrows on the Buparlisib molecular weight cross-sectional profiles indicate the appearance of the groove. Comparison of hillock formation under the constant load Although the friction-induced fabrication can be realized on silicon surfaces with various crystal planes, the friction-induced clonidine hillocks on various silicon crystal planes are a little different, as shown in

Figure 1. To accurately compare the hillock formation on various silicon surfaces, the scratch tests were performed on three silicon crystal planes under the same constant load by AFM both in air and in vacuum. As shown in Figures 2 and 3, the hillocks were created on three silicon crystal planes under a constant load of 50 μN, where the contact pressure was estimated as 8.5 to 10.5 GPa. Figure 2 shows the hillocks produced in air with N of 100 and 200, respectively. Under the same loading condition, the hillock formation was also investigated in vacuum, as shown in Figure 3. Figure 2 AFM images of the friction-induced hillocks on Si(100), Si(110), and Si(111) surfaces produced in air. The F n is 50 μN, and the N is 100 and 200. Figure 3 AFM images of the friction-induced hillocks on Si(100), Si(110), and Si(111) surfaces produced in vacuum. The F n is 50 μN, and the N is 100 and 200. To quantitatively compare the hillock size on various silicon crystal planes, the height and volume of the hillocks were measured with the original silicon surface as the base level.

The specimens were viewed using a Tecnai G2 transmission

electron microscopy at 75 keV. For Western blot, 10 μg purified VLPs were separated by SDS-PAGE electrophoresis and subjected to Western blot assay. (F, H) Electron micrograph images and Western blotting result of VLP H2. (I) RGD-core-IFN-α2a fusion protein bind with breast cancer cells MDA-MB231. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37° under 5% CO2. After 2 h, the cells were washed three times with PBS, and green fluorescence was observed under the fluorescence microscope. Scale bar = 100 μm. Binding specificity assay MDA-MB231 human breast cancer cells were cultured Selleck MK-4827 in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin, at 37°C under 5% CO2. Then, 0.2, 0.5, 2, 5, and 10 μM fusion proteins His-H1, His-H2, His-H3, and His-H4 were co-incubated with MDA-MB231 at 37°C under 5% CO2. After 2 h, the cells were washed click here three times with PBS, and green fluorescence was observed

under the fluorescence microscope. Construction of recombinant baculovirus pH1, pH2, pH3, and pH4 were digested by BamHI/EcoRI and were subcloned into pFastBac dual vector (pFBD) that had been pre-treated with BamHI/EcoRI and produced pFBD-H1, pFBD-H2, pFBD-H3, and pFBD-H4. The four donor plasmids (pFBD-H1, pFBD-H2, pFBD-H3, and pFBD-H4) mediated the insertion of genes into the AcBacmid by Tn7-mediated transposition to generate AcFBD-H1, AcFBD-H2, AcFBD-H3, and AcFBD-H4 bacmids, respectively. These recombinant bacmids were confirmed by PCR and then were introduced by transfection into Sf9 cells to produce the recombinant viruses vAcH1, vAcH2, vAcH3, and vAcH4. Real-time Q-PCR and Western blotting Total

RNA was extracted from cells with PureLink RNA kit (Life Technologies Corporation). cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Thalidomide Biosystems, Foster City, CA, USA). The relative quantification of gene expression for each sample was analyzed by the ΔC t method. The following primers were used to amplify HCV core: 5’- GCC CAC AGG ACG TCA AGT −3’ and 5’- CGC AAC CCT CAT TGC CAT −3’; 18S rRNA: 5’-ACC TGG TTG ATC CTG CCA GT-3’ and 5’-CTG ACC GGG TTG GTT TTG AT-3’. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated on a 12% sodium SB525334 mouse dodecyl sulfate polyacrylamide gel (SDS-PAGE) by electrophoresis and subjected to Western blot assay.