Month: July 2017

E assessment that aquatic JW-74 Viruses are extraordinarily diverse, but the majority of sequences obtainedfrom these investigations are not similar to known genes, indicating that much of the genomic information in aquatic viruses has yet to be characterized 22948146 [10]. The high diversity of aquatic viral communities means that very few sequences from metagenomic analyses can be reassembled into larger stretches of sequence [11?3]. Without reassembly of the fragmented genomes, the genetic structure of individual viruses cannot be assessed and genes cannot be investigated within the context of whole genomes. The current methods used to construct these metagenomic libraries also eliminate any phenotypic information about viruses in the samples. So far, with the exception of a small single-stranded DNA virus [14], reassembly of uncultivated prokaryotic and viral genomes from shotgun libraries of aquatic assemblages has only been achieved with samples that contain low diversity of bacteria or viruses [15?7]. This had led to the suggestion that, in addition to advances in sequencing technology and computational methods [18?0], there should also be a focus on improving upstream methods that are used to prepare samples for metagenomic analyses, specifically methods that reduce the diversity of the samples through physical fractionation [21]. In fact, computational models have shown that separating viruses from a sample into two or more fractions can increase the assembly of sequenced DNA fragments from the constituent viral assemblage [22].Assembly of a Viral Metagenome after FractionationMulti-dimensional physical fractionation of natural aquatic viral assemblages can be achieved by exploiting differences in the sizes, surface charges, and buoyant densities among different populations of viruses [23]. Here, we use two physical fractionation steps in series to enrich a limited number of viral consortia from a complex marine assemblage in order to test whether such a procedure would result in a high proportion of assembled sequences.Materials and Methods Ethics StatementNo specific permits were required for the described field studies. Samples were Castanospermine web collected from public waters and no specific permissions were required. Samples consisted of microscopic plankton, which are not endangered or protected.Sample CollectionA viral concentrate was collected on October 17, 2006 from a depth of 3 m approximately 25 m off the southeast shore of ?Coconut Island (Moku O Lo`e) located 15755315 in Kane`ohe Bay, Oahu, HI. Approximately 1800 l of water was filtered through 0.2 mm pore-size cartridge filters with polyethersulfone membranes (Polycap, Whatman). Viruses in the filtrate were concentrated with a tangential flow filtration cassette with 100 kDa nominal molecular weight cut-off (NMWCO) regenerated cellulose membrane (Pellicon 2, Millipore). The concentrate was stored at 4uC after addition of protease inhibitor (Sigma-Aldrich) at a final concentration of approximately 100 mg l21 in an attempt to decrease viral degradation. The sample was then further concentrated with 100 kDa NMWCO Centricon-80 centrifugal ultrafiltration devices (Millipore) and stored at 4uC until fractionation.Viral Genome Size DistributionsPulsed-field gel electrophoresis (PFGE) was used to monitor viral genome size distributions in the fractions collected from viral fractionation as an indicator of fractionation progress. Viruses in fractions were concentrated with 100 kDa NMWCO Nanosep centrifugal ultr.E assessment that aquatic viruses are extraordinarily diverse, but the majority of sequences obtainedfrom these investigations are not similar to known genes, indicating that much of the genomic information in aquatic viruses has yet to be characterized 22948146 [10]. The high diversity of aquatic viral communities means that very few sequences from metagenomic analyses can be reassembled into larger stretches of sequence [11?3]. Without reassembly of the fragmented genomes, the genetic structure of individual viruses cannot be assessed and genes cannot be investigated within the context of whole genomes. The current methods used to construct these metagenomic libraries also eliminate any phenotypic information about viruses in the samples. So far, with the exception of a small single-stranded DNA virus [14], reassembly of uncultivated prokaryotic and viral genomes from shotgun libraries of aquatic assemblages has only been achieved with samples that contain low diversity of bacteria or viruses [15?7]. This had led to the suggestion that, in addition to advances in sequencing technology and computational methods [18?0], there should also be a focus on improving upstream methods that are used to prepare samples for metagenomic analyses, specifically methods that reduce the diversity of the samples through physical fractionation [21]. In fact, computational models have shown that separating viruses from a sample into two or more fractions can increase the assembly of sequenced DNA fragments from the constituent viral assemblage [22].Assembly of a Viral Metagenome after FractionationMulti-dimensional physical fractionation of natural aquatic viral assemblages can be achieved by exploiting differences in the sizes, surface charges, and buoyant densities among different populations of viruses [23]. Here, we use two physical fractionation steps in series to enrich a limited number of viral consortia from a complex marine assemblage in order to test whether such a procedure would result in a high proportion of assembled sequences.Materials and Methods Ethics StatementNo specific permits were required for the described field studies. Samples were collected from public waters and no specific permissions were required. Samples consisted of microscopic plankton, which are not endangered or protected.Sample CollectionA viral concentrate was collected on October 17, 2006 from a depth of 3 m approximately 25 m off the southeast shore of ?Coconut Island (Moku O Lo`e) located 15755315 in Kane`ohe Bay, Oahu, HI. Approximately 1800 l of water was filtered through 0.2 mm pore-size cartridge filters with polyethersulfone membranes (Polycap, Whatman). Viruses in the filtrate were concentrated with a tangential flow filtration cassette with 100 kDa nominal molecular weight cut-off (NMWCO) regenerated cellulose membrane (Pellicon 2, Millipore). The concentrate was stored at 4uC after addition of protease inhibitor (Sigma-Aldrich) at a final concentration of approximately 100 mg l21 in an attempt to decrease viral degradation. The sample was then further concentrated with 100 kDa NMWCO Centricon-80 centrifugal ultrafiltration devices (Millipore) and stored at 4uC until fractionation.Viral Genome Size DistributionsPulsed-field gel electrophoresis (PFGE) was used to monitor viral genome size distributions in the fractions collected from viral fractionation as an indicator of fractionation progress. Viruses in fractions were concentrated with 100 kDa NMWCO Nanosep centrifugal ultr.

On number NM021175) was cloned from a NT-157 supplier patient liver specimen. In brief, RNA was extracted using TRIzol RNA isolation reagent (Invitrogen). The cDNA was synthesized using RT II reverse transcriptase (Invitrogen), and 2 ml of cDNA was used for PCR amplification. Hepcidin gene was amplified with primers 59-ACCAGAGCAAGCTCAAGACC-39 and 59-CAGGGCAGGTAGGTTCTACG-39. The reaction was performed at 94uC for 3 min, followed by 38 cycles at 94uC for 40 s, 58uC for 30 s, 72uC for 45 s and finished with an extension at 72uC for 10 min. The PCR product was then purified and cloned into pEF6/V5-His-TOPO vector. The expression vector pTOPO-hepcidin and the anti-sense vector pTOPO-anti-hepcidin were sequenced using the BigDye Terminator V3.1 Kit from Applied Biosystems.Small Interfering RNA TransfectionTo knockdown STAT3, synthesized small interfering RNA (siRNA) duplexes were purchased from Cell Signaling co. (Danvers, MA). To knockdown 1326631 hepcidin, plasmid shRNA specific for hepcidin was purchased from Source Bioscience (Nottingham, UK). Cells were seeded in six-well plates 16 hours before transfection at a confluence of 50 , and transfected with 100 nM siRNA or 2 mg plasmid shRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.RNA Extraction, Reverse Transcription-PCR (RT-PCR) and Quantitative Real-time PCRRNA was extracted from cells or patient tissues using TRIzol RNA isolation reagent (Invitrogen, Carlsbad, CA). To prevent DNA contamination, total RNA was treated with RNase-free DNase II (Invitrogen, Carlsbad, CA). Human glyceraldehyde-3phosphate dehydrogenase gene (GAPDH, forward primer 59TCACCAGGGCTGCTTTTA-39 and reverse primer 59-TTCACACCCATGACGAACA-39) was used as an internal control in the PCR amplification. A two-step RT-PCR procedure was performed in all experiments. First, total RNA samples (1.6 mg per reaction) were reversely transcribed into cDNAs by RT II reverse transcriptase (Invitrogen). Then, the cDNAs were used as templates in PCR with hepcidin specific primers 59-ACCAGAGCAAGCTCAAGACC-39 and 59-CAGGGCAGGTAGGTTCTACG-39 or with HCV specific primers 59-TTCACGCAGAAAGCGTCTAG-39 and 59-CACTCGCAAGCACCCTATCAGGCAG-39. The primers for IFIT1 detection were 59TGGCTAAGCAAAACCCTGCA-39 and 59-TCTGGCCTTTCAGGTGTTTCAC-39. The primers for OAS1 detection were 59-AGGTGGTAAAGGGTGGCTCC-39 and 59-ACAACCAGGTCAGCGTCAGAT-39. The amplification reactions were performed by using AmpliTaq Gold (Applied Biosystems, Foster City, CA), and PCR bands were visualized under UV light andWestern Blot AnalysisProteins were separated by SDS-PAGE (10 or 15 acrylamide), transferred to nitrocellulose membranes, and then blocked with 5 skim milk in a phosphate-buffered saline. Membranes were incubated with one of the following antibodies: mouse antihepcidin monoclonal BIBS39 web antibody (1:250; abcam), anti-STAT3 antibody (1:250; Santa Cruz), anti-pSTAT3 antibody (1:100; Santa Cruz), or anti-actin antibody (1:8000; Sigma-Aldrich). After washing, membranes were incubated with peroxidase-conjugated goat anti-mouse immunoglobulin G or goat anti-rabbit immunoglobulin G (Sigma-Aldrich). Signals were detected by using the SupersignalH West Pico Chemiluminescent Substrate (PIERCE) according to the manufacturer’s directions.ImmunofluorescenceCells were grown on glass cover slips and fixed with 5 acetic acid in ethanol. The cells were washed with phosphate-bufferedHepcidin Exhibits Antiviral Activity against HCVsaline and incubated with monoclonal antibody to HCV.On number NM021175) was cloned from a patient liver specimen. In brief, RNA was extracted using TRIzol RNA isolation reagent (Invitrogen). The cDNA was synthesized using RT II reverse transcriptase (Invitrogen), and 2 ml of cDNA was used for PCR amplification. Hepcidin gene was amplified with primers 59-ACCAGAGCAAGCTCAAGACC-39 and 59-CAGGGCAGGTAGGTTCTACG-39. The reaction was performed at 94uC for 3 min, followed by 38 cycles at 94uC for 40 s, 58uC for 30 s, 72uC for 45 s and finished with an extension at 72uC for 10 min. The PCR product was then purified and cloned into pEF6/V5-His-TOPO vector. The expression vector pTOPO-hepcidin and the anti-sense vector pTOPO-anti-hepcidin were sequenced using the BigDye Terminator V3.1 Kit from Applied Biosystems.Small Interfering RNA TransfectionTo knockdown STAT3, synthesized small interfering RNA (siRNA) duplexes were purchased from Cell Signaling co. (Danvers, MA). To knockdown 1326631 hepcidin, plasmid shRNA specific for hepcidin was purchased from Source Bioscience (Nottingham, UK). Cells were seeded in six-well plates 16 hours before transfection at a confluence of 50 , and transfected with 100 nM siRNA or 2 mg plasmid shRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.RNA Extraction, Reverse Transcription-PCR (RT-PCR) and Quantitative Real-time PCRRNA was extracted from cells or patient tissues using TRIzol RNA isolation reagent (Invitrogen, Carlsbad, CA). To prevent DNA contamination, total RNA was treated with RNase-free DNase II (Invitrogen, Carlsbad, CA). Human glyceraldehyde-3phosphate dehydrogenase gene (GAPDH, forward primer 59TCACCAGGGCTGCTTTTA-39 and reverse primer 59-TTCACACCCATGACGAACA-39) was used as an internal control in the PCR amplification. A two-step RT-PCR procedure was performed in all experiments. First, total RNA samples (1.6 mg per reaction) were reversely transcribed into cDNAs by RT II reverse transcriptase (Invitrogen). Then, the cDNAs were used as templates in PCR with hepcidin specific primers 59-ACCAGAGCAAGCTCAAGACC-39 and 59-CAGGGCAGGTAGGTTCTACG-39 or with HCV specific primers 59-TTCACGCAGAAAGCGTCTAG-39 and 59-CACTCGCAAGCACCCTATCAGGCAG-39. The primers for IFIT1 detection were 59TGGCTAAGCAAAACCCTGCA-39 and 59-TCTGGCCTTTCAGGTGTTTCAC-39. The primers for OAS1 detection were 59-AGGTGGTAAAGGGTGGCTCC-39 and 59-ACAACCAGGTCAGCGTCAGAT-39. The amplification reactions were performed by using AmpliTaq Gold (Applied Biosystems, Foster City, CA), and PCR bands were visualized under UV light andWestern Blot AnalysisProteins were separated by SDS-PAGE (10 or 15 acrylamide), transferred to nitrocellulose membranes, and then blocked with 5 skim milk in a phosphate-buffered saline. Membranes were incubated with one of the following antibodies: mouse antihepcidin monoclonal antibody (1:250; abcam), anti-STAT3 antibody (1:250; Santa Cruz), anti-pSTAT3 antibody (1:100; Santa Cruz), or anti-actin antibody (1:8000; Sigma-Aldrich). After washing, membranes were incubated with peroxidase-conjugated goat anti-mouse immunoglobulin G or goat anti-rabbit immunoglobulin G (Sigma-Aldrich). Signals were detected by using the SupersignalH West Pico Chemiluminescent Substrate (PIERCE) according to the manufacturer’s directions.ImmunofluorescenceCells were grown on glass cover slips and fixed with 5 acetic acid in ethanol. The cells were washed with phosphate-bufferedHepcidin Exhibits Antiviral Activity against HCVsaline and incubated with monoclonal antibody to HCV.

N maters Title Loaded From File relative to non-maters, we speculate that structural differences in synaptic morphology are involved. However, further studies investigating the exact relationship between increased levels of tau and 1317923 relative increased dendritic spine density are necessary before a causal link can be established. Given declining libido during ageing in men, these studies could lead to discoveries with translational implications.AcknowledgmentsWe would like to thank Aileen Wills and Elizabeth Boates for technical assistance.Author ContributionsConceived and designed the experiments: JHP EFR. Performed the experiments: PB CM AMKM PJB SZ JHP. Analyzed the data: JHP. Contributed reagents/materials/analysis tools: JHP EFR SZ. Wrote the paper: PB CM EFR JHP.
Several morphological and physiological changes occur in the mammary gland during the processes of gestation and lactation [1?]. During gestation, there is an In the lung.Materials and Methods SubjectsA total of 296 patients with increase in the formation of the epithelial cells, which are involved in milk synthesis, from mammary fat cells [5]. During this stage, several hormones are involved in the regulation of the expression of specific genes to prepare the mammary gland for a successful lactation period [6?12]. During lactation, there is a sharp increase in the synthesis of the components of the milk, particularly proteins, lipids and lactose, in the epithelial cells in the mammary gland [13]. To sustain all the metabolic adaptations that occur during gestation and lactation in the mammary gland, the supply of nutrients to the dam is essential. It has been demonstrated that food restriction during these periods can modify the outcome of lactation. Food deprivation or the consumption of a low-energy diet during gestation and lactation has been shown to reduce or stop milk synthesis and secretion [14?6]. Therefore, the amount and quality of the diet have profound effects on milk synthesis [17]. In addition to the diet, during short periods of time, the bodyorgans of the dam can also supply nutrients to the mammary gland for the differentiation of cells during gestation, mainly for the synthesis of milk components during lactation. It is known that the liver and the adipose tissue can actively participate in the supply of nutrients to the mammary gland [18?2]. To prepare the mammary gland for gestation and lactation, it is known that the regulation of the expression of genes coding for the transcription factors and enzymes involved in anabolic and catabolic processes is required [13,23?7]. In particular, these include genes involved in lipogenesis (SREBP-1c and fatty acid synthase FAS) [28], protein synthesis (mTOR) [29], glyceroneogenesis (PEPCK) [30], and fatty acid oxidation (CPT-1) [26]. In addition, the supply of nutrients may also regulate the phosphorylation state of proteins involved in the activation of protein synthesis (S6K) via mTOR [31,32] and the energy status of the cell via adenosine monophosphate kinase (AMPK) [33,34]. The correct activation of these pathways leads to optimal milk synthesis and secretion. This has been confirmed by numerous studies that have demonstrated that these and other genes are actively regulated during the gestation and lactation stages [13,23?7]. In addition, the expression of several of these genes is regulated inDietary Protein and Mammary Gland MetabolismTable 1. Composition of the experimental diets used in this study.Ingredients g/kg diet CaseinaPercentage ( ) of dietary protein/dietary carbohydrates 10/73 100.0 152.5.N maters relative to non-maters, we speculate that structural differences in synaptic morphology are involved. However, further studies investigating the exact relationship between increased levels of tau and 1317923 relative increased dendritic spine density are necessary before a causal link can be established. Given declining libido during ageing in men, these studies could lead to discoveries with translational implications.AcknowledgmentsWe would like to thank Aileen Wills and Elizabeth Boates for technical assistance.Author ContributionsConceived and designed the experiments: JHP EFR. Performed the experiments: PB CM AMKM PJB SZ JHP. Analyzed the data: JHP. Contributed reagents/materials/analysis tools: JHP EFR SZ. Wrote the paper: PB CM EFR JHP.
Several morphological and physiological changes occur in the mammary gland during the processes of gestation and lactation [1?]. During gestation, there is an increase in the formation of the epithelial cells, which are involved in milk synthesis, from mammary fat cells [5]. During this stage, several hormones are involved in the regulation of the expression of specific genes to prepare the mammary gland for a successful lactation period [6?12]. During lactation, there is a sharp increase in the synthesis of the components of the milk, particularly proteins, lipids and lactose, in the epithelial cells in the mammary gland [13]. To sustain all the metabolic adaptations that occur during gestation and lactation in the mammary gland, the supply of nutrients to the dam is essential. It has been demonstrated that food restriction during these periods can modify the outcome of lactation. Food deprivation or the consumption of a low-energy diet during gestation and lactation has been shown to reduce or stop milk synthesis and secretion [14?6]. Therefore, the amount and quality of the diet have profound effects on milk synthesis [17]. In addition to the diet, during short periods of time, the bodyorgans of the dam can also supply nutrients to the mammary gland for the differentiation of cells during gestation, mainly for the synthesis of milk components during lactation. It is known that the liver and the adipose tissue can actively participate in the supply of nutrients to the mammary gland [18?2]. To prepare the mammary gland for gestation and lactation, it is known that the regulation of the expression of genes coding for the transcription factors and enzymes involved in anabolic and catabolic processes is required [13,23?7]. In particular, these include genes involved in lipogenesis (SREBP-1c and fatty acid synthase FAS) [28], protein synthesis (mTOR) [29], glyceroneogenesis (PEPCK) [30], and fatty acid oxidation (CPT-1) [26]. In addition, the supply of nutrients may also regulate the phosphorylation state of proteins involved in the activation of protein synthesis (S6K) via mTOR [31,32] and the energy status of the cell via adenosine monophosphate kinase (AMPK) [33,34]. The correct activation of these pathways leads to optimal milk synthesis and secretion. This has been confirmed by numerous studies that have demonstrated that these and other genes are actively regulated during the gestation and lactation stages [13,23?7]. In addition, the expression of several of these genes is regulated inDietary Protein and Mammary Gland MetabolismTable 1. Composition of the experimental diets used in this study.Ingredients g/kg diet CaseinaPercentage ( ) of dietary protein/dietary carbohydrates 10/73 100.0 152.5.

Howing GFP expression viewed using phase-contrast optics (left) or the same field under fluorescence illumination (right). doi:10.1371/journal.pone.0064613.gGene Attenuation in Cloned PigsProduction of Cloned Pigs from shRNA1 Transfected Fibroblast CellsThe transfer of 284 cloned embryos reconstructed from transfected fibroblasts to five recipient gilts resulted in the birth of 8 cloned piglets from 3 gilts (4, 2 and 2 piglets, respectively). One of the piglets (1 of 4) from one of the recipient gilt was stillborn. The remaining 7 piglets were healthy and had normal morphology at birth and weaning (Figure 4A). One piglet from another recipient sow died of a respiratory infection after weaning at age of 4 weeks. All the other surviving clone pigs had normal growth and were healthy until they were euthanized. GFP was detected in tissue samples collected from all the apoE-shRNA1 transgenic cloned pigs but not in Title Loaded From File tissues of control cloned pigs (Figure 4B). This indicated the stable integration of the apoEshRNA1 vector and transgene expression in the tissues of the transgenic pigs. PCR analysis of genomic DNA extracted from liver samples confirmed the presence of the apoE-shRNA1 vector in the genome of pigs cloned from apoE-shRNA1 fibroblasts cells but not in the genome of pigs cloned from non-transfected control fibroblasts (Figure 4C).(Figures 5A and 6A). However, densitometric analysis of the protein bands after immunoblotting revealed lower levels of apoE in both liver (Figure 5B) and plasma (Figure 6B) of cloned transgenic pigs as compared to control pigs. Immunoblot analyses of liver samples using an anti-GFP antibody confirmed that GFP was highly expressed in transgenic pigs (Figure 5A).DiscussionThere is great promise in the use of genetically-modified swine to improve our understanding of biology and diseases. Indeed, because swine are anatomically and physiologically Title Loaded From File similar to humans, the alteration of specific swine genes can provide ideal animal models to study the causes and potential therapeutics of genetic disorders affecting humans [2]. The swine genome is now sequenced and will facilitate the design and creation of geneticallyaltered swine models [25]. However, in order to enable the adoption of swine models in biomedical applications, the methods of gene manipulation as well as in the technologies used to produce gene-altered pigs require further refinements to improve efficiency, precision and simplicity. Therefore, the primary goal of this study was to determine the feasibility of using RNAi to modify gene expression in tissues and plasma of cloned pigs. RNAi is a natural gene silencing mechanism triggered by double stranded RNA, which is highly conserved among different species [26]. The fact that stable gene silencing can be achieved by short hairpin RNAs (shRNA) expressed from DNA vectors via polymerase III promoters [27?9] has provided an appealing alternative to the conventional methods for gene targeting in animals [17,18,20,21]. The shRNA consists of a sense and antisense small interfering RNA (siRNA) sequences linked by a non-complementary loop sequence. Upon expression, the loop isDetection of apoE Protein in the Cloned PigsIn order to assess whether the presence of the apoE-shRNA1 vector affected the levels of the apoE protein, liver and plasma samples collected from the transgenic clone pigs and control clone pigs were analyzed. ApoE protein was detected in all liver and plasma samples from both control and tr.Howing GFP expression viewed using phase-contrast optics (left) or the same field under fluorescence illumination (right). doi:10.1371/journal.pone.0064613.gGene Attenuation in Cloned PigsProduction of Cloned Pigs from shRNA1 Transfected Fibroblast CellsThe transfer of 284 cloned embryos reconstructed from transfected fibroblasts to five recipient gilts resulted in the birth of 8 cloned piglets from 3 gilts (4, 2 and 2 piglets, respectively). One of the piglets (1 of 4) from one of the recipient gilt was stillborn. The remaining 7 piglets were healthy and had normal morphology at birth and weaning (Figure 4A). One piglet from another recipient sow died of a respiratory infection after weaning at age of 4 weeks. All the other surviving clone pigs had normal growth and were healthy until they were euthanized. GFP was detected in tissue samples collected from all the apoE-shRNA1 transgenic cloned pigs but not in tissues of control cloned pigs (Figure 4B). This indicated the stable integration of the apoEshRNA1 vector and transgene expression in the tissues of the transgenic pigs. PCR analysis of genomic DNA extracted from liver samples confirmed the presence of the apoE-shRNA1 vector in the genome of pigs cloned from apoE-shRNA1 fibroblasts cells but not in the genome of pigs cloned from non-transfected control fibroblasts (Figure 4C).(Figures 5A and 6A). However, densitometric analysis of the protein bands after immunoblotting revealed lower levels of apoE in both liver (Figure 5B) and plasma (Figure 6B) of cloned transgenic pigs as compared to control pigs. Immunoblot analyses of liver samples using an anti-GFP antibody confirmed that GFP was highly expressed in transgenic pigs (Figure 5A).DiscussionThere is great promise in the use of genetically-modified swine to improve our understanding of biology and diseases. Indeed, because swine are anatomically and physiologically similar to humans, the alteration of specific swine genes can provide ideal animal models to study the causes and potential therapeutics of genetic disorders affecting humans [2]. The swine genome is now sequenced and will facilitate the design and creation of geneticallyaltered swine models [25]. However, in order to enable the adoption of swine models in biomedical applications, the methods of gene manipulation as well as in the technologies used to produce gene-altered pigs require further refinements to improve efficiency, precision and simplicity. Therefore, the primary goal of this study was to determine the feasibility of using RNAi to modify gene expression in tissues and plasma of cloned pigs. RNAi is a natural gene silencing mechanism triggered by double stranded RNA, which is highly conserved among different species [26]. The fact that stable gene silencing can be achieved by short hairpin RNAs (shRNA) expressed from DNA vectors via polymerase III promoters [27?9] has provided an appealing alternative to the conventional methods for gene targeting in animals [17,18,20,21]. The shRNA consists of a sense and antisense small interfering RNA (siRNA) sequences linked by a non-complementary loop sequence. Upon expression, the loop isDetection of apoE Protein in the Cloned PigsIn order to assess whether the presence of the apoE-shRNA1 vector affected the levels of the apoE protein, liver and plasma samples collected from the transgenic clone pigs and control clone pigs were analyzed. ApoE protein was detected in all liver and plasma samples from both control and tr.

Igand pose program and ranked by Consensus score program. Finally, the optimal 3D binding conformations of complexes were selected and shown in Figure 7. The interaction energies between key residues and the ligand are listed in the Table 1. All of these residues are located in the cavity formed by six helices. Several residues including Ile45, Leu49, Thr64, Leu68 and Leu82 identified by current docking simulations have been shown to play an important role in the binding of CSPSlit and rhodojaponin III. Figure 8 shows the time series of potential energy and RMSD of backbone for 1000 ps MD simulation of CSPSlit structure. The potential energy of the complex was stabilized at 200 ps production after 800 ps equilibration, and the RMSD of backbone compared to the ?starting coordinate remained at 0.14 A up and down fluctuations. These two properties converged at production, indicating that the complex is stable.Table 1. VdW Energy (EvdW) and Electrostatic Energy (Eele) between rhodojaponin III and CSPSlit.Residue Leu41 Ile45 Ala48 Leu49 Cys53 Cys56 Gln60 Thr64 Val67 Leu68 Leu71 Trp79 Leu82 Cys83 Tyr86 Asp87 TyrEvdW (kcal/mol) 20.830793 23.700930 21.080190 22.234000 20.358571 20.318546 21.109890 22.165880 22.477020 22.773280 20.885637 0.174320 22.684090 21.524360 21.640490 20.697000 20.Eele (kcal/mol) 0.017039 20.346153 0.739731 20.098858 0.154808 20.240461 0.331317 20.800064 0.549604 20.523853 0.179657 0.187195 0.219521 0.297607 0.447274 20.607238 0.3.5 Binding Assay of Rhodojaponin III and CSPSlitPurified CSPSlit protein solution contained 5.0 mg/ml was used to analyse the binding property of CSPSlit with rhodojaponin III. When excited at 295 nm, the fluorescence emission spectra showed maximally relative fluorescence intensity at 383.6 nm for CSPSlit. Following, with the increasing concentration of rhodojaponin III, CSPSlit peak underwent a blue shift, but no peak intensity increaseing was observed. When the final concentration of rhodojaponin III was 600 mM and 300 mM, the fluorescence intensity decreased to 29.43 and 18.47 , respectively (Fig. 9). These results showed that CSPSlit could be intensely bound with rhodojaponin III.DiscussionThe olfactory system of insects is essential for Lepidoptera as well as in other insect orders to initiate behavioral responses, suchdoi:10.1371/journal.pone.0047611.tCharacterisation Binding Properties of CSPSlitFigure 8. Potential energy (A) and root-mean-square deviation (B) with respect to simulation time for 1000 ps MedChemExpress NT-157 molecular dynamics simulation on the CSPSlit- rhodojaponin III complex model. doi:10.1371/journal.pone.0047611.gas searching for food sources, mating, oviposition and feeding [52?53]. Chemosensory proteins (CSPs), known as another class of soluble protein, share no sequence homology with either PBPs or general OBPs of many insects [11?2,18]. The CSPs are smaller proteins which contain four cysteines instead of six with 57773-63-4 site conserved interval spacing involved in two disulfide bonds [35,54]. In the present study, a cDNA sequence encoding the CSP of S. litura was cloned. The CSPSlit has 4 typical conservative cysteines in the sequence (Fig.1). It is consistent with previous report. The CSPSlit was expressed in antennae, legs, wings and female abdomens (Fig.3), these results is similar with the research in other insect [9,21]. In M. brassicae, the CSPs has a abundant expression in proboscis [15,30], but in this study, there is no hybridization signal was detected in the de-antennated head (Fig.Igand pose program and ranked by Consensus score program. Finally, the optimal 3D binding conformations of complexes were selected and shown in Figure 7. The interaction energies between key residues and the ligand are listed in the Table 1. All of these residues are located in the cavity formed by six helices. Several residues including Ile45, Leu49, Thr64, Leu68 and Leu82 identified by current docking simulations have been shown to play an important role in the binding of CSPSlit and rhodojaponin III. Figure 8 shows the time series of potential energy and RMSD of backbone for 1000 ps MD simulation of CSPSlit structure. The potential energy of the complex was stabilized at 200 ps production after 800 ps equilibration, and the RMSD of backbone compared to the ?starting coordinate remained at 0.14 A up and down fluctuations. These two properties converged at production, indicating that the complex is stable.Table 1. VdW Energy (EvdW) and Electrostatic Energy (Eele) between rhodojaponin III and CSPSlit.Residue Leu41 Ile45 Ala48 Leu49 Cys53 Cys56 Gln60 Thr64 Val67 Leu68 Leu71 Trp79 Leu82 Cys83 Tyr86 Asp87 TyrEvdW (kcal/mol) 20.830793 23.700930 21.080190 22.234000 20.358571 20.318546 21.109890 22.165880 22.477020 22.773280 20.885637 0.174320 22.684090 21.524360 21.640490 20.697000 20.Eele (kcal/mol) 0.017039 20.346153 0.739731 20.098858 0.154808 20.240461 0.331317 20.800064 0.549604 20.523853 0.179657 0.187195 0.219521 0.297607 0.447274 20.607238 0.3.5 Binding Assay of Rhodojaponin III and CSPSlitPurified CSPSlit protein solution contained 5.0 mg/ml was used to analyse the binding property of CSPSlit with rhodojaponin III. When excited at 295 nm, the fluorescence emission spectra showed maximally relative fluorescence intensity at 383.6 nm for CSPSlit. Following, with the increasing concentration of rhodojaponin III, CSPSlit peak underwent a blue shift, but no peak intensity increaseing was observed. When the final concentration of rhodojaponin III was 600 mM and 300 mM, the fluorescence intensity decreased to 29.43 and 18.47 , respectively (Fig. 9). These results showed that CSPSlit could be intensely bound with rhodojaponin III.DiscussionThe olfactory system of insects is essential for Lepidoptera as well as in other insect orders to initiate behavioral responses, suchdoi:10.1371/journal.pone.0047611.tCharacterisation Binding Properties of CSPSlitFigure 8. Potential energy (A) and root-mean-square deviation (B) with respect to simulation time for 1000 ps molecular dynamics simulation on the CSPSlit- rhodojaponin III complex model. doi:10.1371/journal.pone.0047611.gas searching for food sources, mating, oviposition and feeding [52?53]. Chemosensory proteins (CSPs), known as another class of soluble protein, share no sequence homology with either PBPs or general OBPs of many insects [11?2,18]. The CSPs are smaller proteins which contain four cysteines instead of six with conserved interval spacing involved in two disulfide bonds [35,54]. In the present study, a cDNA sequence encoding the CSP of S. litura was cloned. The CSPSlit has 4 typical conservative cysteines in the sequence (Fig.1). It is consistent with previous report. The CSPSlit was expressed in antennae, legs, wings and female abdomens (Fig.3), these results is similar with the research in other insect [9,21]. In M. brassicae, the CSPs has a abundant expression in proboscis [15,30], but in this study, there is no hybridization signal was detected in the de-antennated head (Fig.

He figure also illustrated a longer exponential growth phase to a higher OD600 value (0.19) than the control (0.10). It was noted that the deletion of crp (JW5702 Dkan+blank plasmid pKSC) did not improve the ethanol tolerance of E. coli.Chromosomal IntegrationSince E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Dkan, the native crp operon location, to create Pentagastrin site strain iE2. Chromosomal integration was performed to avoid the disadvantages of using plasmid-based system, such as genetic instability arising from segregation or horizontal gene transfer, metabolic burden, and the need for supplementing antibiotics [44,45]. DNA sequencing results confirmed the same amino acid substitution in the crp operon of iE2 as E2. The growth of iE2 under ethanol stress was investigated with its parent strain BW25113 and JW5702. In the absence of ethanol, iE2 shared similar growth pattern with BW25113, with a growth rate 25331948 around 0.45 h21, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h21) not only outgrew BW25113 (0.055 h21) but also reached a higher OD600 value (0.13) than BW25113 (0.09) after 12 h (Figure 2B). The crp knock-out strain JW5702 exhibited the worst ethanol tolerance among all three strains. It was also noted that when ethanol concentration was low, iE2 might result in worse growth than the parent strain (data not shown). To further prove the ethanol tolerance of iE2, both iE2 and BW25113 were exposed to 150 g/l ethanol and their survival was recorded over time (Figure 3). iE2 exhibited significantly better survival than BW25113 over the 1-h period examined. For instance, after 15-min exposure to 150 g/l ethanol, iE2 displayed more than 10 survival whereas BW25113 only had less than 0.01 . Even after 1-h exposure, iE2 still demonstrated 15900046 over 10,000-fold survival than BW25113.Cross Resistance to other AlcoholsCell culture was prepared by diluting overnight seed into fresh LB medium containing different alcohols. Cell growth was recorded by OD600 readings with the incubation at 37uC, 200 rpm. The alcohol concentrations used are presented in volume ratio: 3.1 1-propanol, 1.3 1-butanol, and 0.45 1pentanol.Results Isolation of Ethanol-tolerant CRP MutantsError-prone PCR was carried out to introduce 2? nucleotide substitutions per crp by varying the amount of DNA template. Recombinant CASIN plasmids with mutated crp inserts were transformed into competent E. coli JW5702 Dkan (crp knock-out strain) and the total error-prone library size was greater than 106. The mutagenesis libraries were then enriched through repeated subcultures containing 40?5 g/l ethanol to separate “winners” with enhanced ethanol tolerance. The mutated crp inserts of these “winners” were digested, re-ligated to freshly prepared plasmid pKSC, and the resulting recombinant plasmids were re-transformed into E. coli JW5702 Dkan background to eliminate false positives or chromosomal mutations. Three ethanol-tolerant mutants (E1 3) with improved growth under ethanol stress were selected and their amino acid substitutions are summarized in Table 2.Resistance towards other AlcoholsThe tolerance ability of iE2 towards other alcohols, namely 1propanol, 1-butanol, and 1-pentanol, was also studied to demonstrate its alcohol tolerance in general (Figure 4). iE2 showed much better growth than BW25113 and was able to achieve a higher.He figure also illustrated a longer exponential growth phase to a higher OD600 value (0.19) than the control (0.10). It was noted that the deletion of crp (JW5702 Dkan+blank plasmid pKSC) did not improve the ethanol tolerance of E. coli.Chromosomal IntegrationSince E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Dkan, the native crp operon location, to create strain iE2. Chromosomal integration was performed to avoid the disadvantages of using plasmid-based system, such as genetic instability arising from segregation or horizontal gene transfer, metabolic burden, and the need for supplementing antibiotics [44,45]. DNA sequencing results confirmed the same amino acid substitution in the crp operon of iE2 as E2. The growth of iE2 under ethanol stress was investigated with its parent strain BW25113 and JW5702. In the absence of ethanol, iE2 shared similar growth pattern with BW25113, with a growth rate 25331948 around 0.45 h21, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h21) not only outgrew BW25113 (0.055 h21) but also reached a higher OD600 value (0.13) than BW25113 (0.09) after 12 h (Figure 2B). The crp knock-out strain JW5702 exhibited the worst ethanol tolerance among all three strains. It was also noted that when ethanol concentration was low, iE2 might result in worse growth than the parent strain (data not shown). To further prove the ethanol tolerance of iE2, both iE2 and BW25113 were exposed to 150 g/l ethanol and their survival was recorded over time (Figure 3). iE2 exhibited significantly better survival than BW25113 over the 1-h period examined. For instance, after 15-min exposure to 150 g/l ethanol, iE2 displayed more than 10 survival whereas BW25113 only had less than 0.01 . Even after 1-h exposure, iE2 still demonstrated 15900046 over 10,000-fold survival than BW25113.Cross Resistance to other AlcoholsCell culture was prepared by diluting overnight seed into fresh LB medium containing different alcohols. Cell growth was recorded by OD600 readings with the incubation at 37uC, 200 rpm. The alcohol concentrations used are presented in volume ratio: 3.1 1-propanol, 1.3 1-butanol, and 0.45 1pentanol.Results Isolation of Ethanol-tolerant CRP MutantsError-prone PCR was carried out to introduce 2? nucleotide substitutions per crp by varying the amount of DNA template. Recombinant plasmids with mutated crp inserts were transformed into competent E. coli JW5702 Dkan (crp knock-out strain) and the total error-prone library size was greater than 106. The mutagenesis libraries were then enriched through repeated subcultures containing 40?5 g/l ethanol to separate “winners” with enhanced ethanol tolerance. The mutated crp inserts of these “winners” were digested, re-ligated to freshly prepared plasmid pKSC, and the resulting recombinant plasmids were re-transformed into E. coli JW5702 Dkan background to eliminate false positives or chromosomal mutations. Three ethanol-tolerant mutants (E1 3) with improved growth under ethanol stress were selected and their amino acid substitutions are summarized in Table 2.Resistance towards other AlcoholsThe tolerance ability of iE2 towards other alcohols, namely 1propanol, 1-butanol, and 1-pentanol, was also studied to demonstrate its alcohol tolerance in general (Figure 4). iE2 showed much better growth than BW25113 and was able to achieve a higher.

O-face manner, and every structural feature or interaction is repeated twice. It was pointed out by Monod et al. [10] that the effect of a single mutation in complexes with the close-packed form may be much greater than in complexes without dihedral symmetry. This effect may allow such complexes to evolve more readily by the efficient generation of favorable interactions, and this prediction has been supported by recent docking-simulation studies [11?3].In contrast, less attention has been paid to the minor population of ring oligomers having simple n-fold rotational symmetry (designated Cn; Figure 22948146 1A). In our statistical analysis of the PDB, we found that such ring complexes may contain even or odd numbers of subunits, and there is no bias toward even numbers (Figure 1D). Ring-shaped oligomers have a wide variety of symmetry. Prime numbers of subunits give the “lowest” symmetry, and highly composite numbers having many divisors (such as 6 and 12) give the “highest” symmetry. A question then arises whether there is a biological or physical reason for rings to evolve with a prime number or highly composite number of subunits. To answer this question, we studied trp RNA binding attenuation protein (TRAP) as an illustrative case. TRAP is a ring-form homooligomer for which crystal structures are available of get BIBS39 11-mer (prime number) and 12-mer (highly composite number) forms (Figure 2A and B). TRAP is found in various species of Bacillus, and plays a central role in the regulation of transcription and 1662274 translation of the trp operon [14]. The monomers of TRAP form a ring-form homo 11-mer with a minor component of 12-mer depending on the solution conditions [16?7]. Each subunit of TRAP is composed of seven-stranded anti-parallel b-sheets and a bound tryptophan molecule. Benzocaine Recently, Tame et al. solved the crystal structure of 12-mer TRAP, which was produced artificially by joining the subunits of B. stearothermophilus TRAP in tandem with linkers of alanine residues [18,19] (Figure 2B). The crystal structure of 12mer TRAP shows exactly the same hydrogen bonding pattern and buried surface as those of the wild-type 11-mer TRAP. Allatom root mean square displacement (RMSD) between theInfluence of Symmetry on Protein DynamicsFigure 1. Ring and close-packed forms. (A) A schematic representation of a ring shaped oligomer. Subunits are arranged symmetrically (Cn symmetry) around the rotational axis (axis 1). Color gradation indicates the top and bottom of the subunit. (B) Schematic representation of a closepacked oligomer. The oligomer composed of n subunits has n/2-fold rotational symmetry around the axis 1, and 2-fold rotational symmetry around each of axes 2?. (C) The number of homooligomers (see Materials and Methods in detail). (D) The number of ring-shaped oligomers. doi:10.1371/journal.pone.0050011.g?monomer of the 11-mer and that of 12-mer was only 0.26 A (Figure 2C and D). Despite their structural similarity, however, 12-mer TRAP is significantly less stable, as shown from the population of 12-mer in solution [15?7]. In this study, we tried to address the influence of the differences in symmetry on the dynamics of the oligomers. The 12-mer structure was modeled with subunits carrying no peptide linkers to stabilize the 12-mer form. We performed 100 ns fully-atomistic MD simulations with an explicit water environment for both forms of TRAPs as well as normal mode analysis using an elastic network model (ENM) [20,21]. The normal mode analysis wit.O-face manner, and every structural feature or interaction is repeated twice. It was pointed out by Monod et al. [10] that the effect of a single mutation in complexes with the close-packed form may be much greater than in complexes without dihedral symmetry. This effect may allow such complexes to evolve more readily by the efficient generation of favorable interactions, and this prediction has been supported by recent docking-simulation studies [11?3].In contrast, less attention has been paid to the minor population of ring oligomers having simple n-fold rotational symmetry (designated Cn; Figure 22948146 1A). In our statistical analysis of the PDB, we found that such ring complexes may contain even or odd numbers of subunits, and there is no bias toward even numbers (Figure 1D). Ring-shaped oligomers have a wide variety of symmetry. Prime numbers of subunits give the “lowest” symmetry, and highly composite numbers having many divisors (such as 6 and 12) give the “highest” symmetry. A question then arises whether there is a biological or physical reason for rings to evolve with a prime number or highly composite number of subunits. To answer this question, we studied trp RNA binding attenuation protein (TRAP) as an illustrative case. TRAP is a ring-form homooligomer for which crystal structures are available of 11-mer (prime number) and 12-mer (highly composite number) forms (Figure 2A and B). TRAP is found in various species of Bacillus, and plays a central role in the regulation of transcription and 1662274 translation of the trp operon [14]. The monomers of TRAP form a ring-form homo 11-mer with a minor component of 12-mer depending on the solution conditions [16?7]. Each subunit of TRAP is composed of seven-stranded anti-parallel b-sheets and a bound tryptophan molecule. Recently, Tame et al. solved the crystal structure of 12-mer TRAP, which was produced artificially by joining the subunits of B. stearothermophilus TRAP in tandem with linkers of alanine residues [18,19] (Figure 2B). The crystal structure of 12mer TRAP shows exactly the same hydrogen bonding pattern and buried surface as those of the wild-type 11-mer TRAP. Allatom root mean square displacement (RMSD) between theInfluence of Symmetry on Protein DynamicsFigure 1. Ring and close-packed forms. (A) A schematic representation of a ring shaped oligomer. Subunits are arranged symmetrically (Cn symmetry) around the rotational axis (axis 1). Color gradation indicates the top and bottom of the subunit. (B) Schematic representation of a closepacked oligomer. The oligomer composed of n subunits has n/2-fold rotational symmetry around the axis 1, and 2-fold rotational symmetry around each of axes 2?. (C) The number of homooligomers (see Materials and Methods in detail). (D) The number of ring-shaped oligomers. doi:10.1371/journal.pone.0050011.g?monomer of the 11-mer and that of 12-mer was only 0.26 A (Figure 2C and D). Despite their structural similarity, however, 12-mer TRAP is significantly less stable, as shown from the population of 12-mer in solution [15?7]. In this study, we tried to address the influence of the differences in symmetry on the dynamics of the oligomers. The 12-mer structure was modeled with subunits carrying no peptide linkers to stabilize the 12-mer form. We performed 100 ns fully-atomistic MD simulations with an explicit water environment for both forms of TRAPs as well as normal mode analysis using an elastic network model (ENM) [20,21]. The normal mode analysis wit.

He supernatant play an important role for the pathogenicity of the bacteria. Previous literature showed that the b-hemolysin contributes to the stimulation of IL-8 release [8] [16]. Because a high IL-8 release from THP-1 macrophages was also observed following stimulation with strain BSU 98 as well as the nonThe GBS ?Hemolysin and Intracellular SurvivalFigure 3. Effect of Cytochalasin D on invasion capacity of S. agalactiae in macrophages. Cytochalasin D treated THP-1 macrophages were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria for 90 min followed by adding antibiotics for 1 h 15900046 to kill extracellular bacteria. Infected THP-1 macrophages without Cytochalasin D treatment served as positive control. Data shown are the mean 6 SD of six independent experiments. Data is considered significant for p values ,0.05 (*) and highly significant for p values ,0.01 (**). doi:10.1371/journal.pone.0060160.ghemolytic strain BSU 453 (Fig. 6A), we hypothesize that the presence of b-hemolysin in BSU 98 may not be the sole mediator for IL-8 release, suggesting the involvement of other bacterial factors that could trigger the release of proinflammatory cytokines. The bacterial cell wall components of gram positive bacteria are a major proinflammatory stimulus and trigger the innate immune system similarly to lipopolysaccharide (LPS) of gram negative bacteria [15] [18]. To investigate the role of cell wall, THP-1 macrophages were stimulated with S. agalactiae cell wall preparations from hemolytic and nonhemolytic strains. A dose and time dependency was observed in the buy KS 176 production of IL-8 following stimulation with 0.1, 1, 5 and 10 mg/ml of cell wall preparations. Maximum stimulation was observed for 1 mg of LPS that served as a positive control in the assay. As b-hemolysin activity is lost during the cell wall isolation method, the resultant cell wall fragments from BSU 6 and BSU 281 show a similar response towards the production of IL-8 as shown in Fig. 7A and 7B.DiscussionSevere invasive S. agalactiae infections are not only a major cause of infections in newborns but also in nonpregnant adults. Bacterial invasion and disease pathogenesis is a complex process that is achieved through numerous virulence factors. The S. agalactiae bhemolysin is considered as one of the most important virulence factors in this context. Invasive S. agalactiae infections are almost exclusively caused by b-hemolytic strains. However, the role of the S. agalactiae b-hemolysin in the molecular interaction with host cells is not completely understood.MedChemExpress IQ1 Interestingly, the cov (or csr) system as a major regulator of S. agalactiae virulence genes suppresses b-hemolysin expression [19] [9]. A recent study by Sendi et al. found that a strain carrying a cov mutation resulting in a high hemolytic S. agalactiae variant with low capsule expression showed low intracellular survival in human neutrophils in contrast to the low hemolytic variant with high capsule expression [8]. To elucidate whether the observed difference is due to b-hemolysin or capsule expression, we used a serotype Ia strain and its isogenic nonhemolytic mutant to investigate the potential factors responsible for the different survival in eukaryotic host cells. The b-hemolytic S. agalactiae wild type strain was found in lower numbers in the intracellular compartment of THP-1 macrophages in comparison to the nonhemolytic mutant strain. With increasing incubation time (from 0.75 to 1.5 h), the number of reco.He supernatant play an important role for the pathogenicity of the bacteria. Previous literature showed that the b-hemolysin contributes to the stimulation of IL-8 release [8] [16]. Because a high IL-8 release from THP-1 macrophages was also observed following stimulation with strain BSU 98 as well as the nonThe GBS ?Hemolysin and Intracellular SurvivalFigure 3. Effect of Cytochalasin D on invasion capacity of S. agalactiae in macrophages. Cytochalasin D treated THP-1 macrophages were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria for 90 min followed by adding antibiotics for 1 h 15900046 to kill extracellular bacteria. Infected THP-1 macrophages without Cytochalasin D treatment served as positive control. Data shown are the mean 6 SD of six independent experiments. Data is considered significant for p values ,0.05 (*) and highly significant for p values ,0.01 (**). doi:10.1371/journal.pone.0060160.ghemolytic strain BSU 453 (Fig. 6A), we hypothesize that the presence of b-hemolysin in BSU 98 may not be the sole mediator for IL-8 release, suggesting the involvement of other bacterial factors that could trigger the release of proinflammatory cytokines. The bacterial cell wall components of gram positive bacteria are a major proinflammatory stimulus and trigger the innate immune system similarly to lipopolysaccharide (LPS) of gram negative bacteria [15] [18]. To investigate the role of cell wall, THP-1 macrophages were stimulated with S. agalactiae cell wall preparations from hemolytic and nonhemolytic strains. A dose and time dependency was observed in the production of IL-8 following stimulation with 0.1, 1, 5 and 10 mg/ml of cell wall preparations. Maximum stimulation was observed for 1 mg of LPS that served as a positive control in the assay. As b-hemolysin activity is lost during the cell wall isolation method, the resultant cell wall fragments from BSU 6 and BSU 281 show a similar response towards the production of IL-8 as shown in Fig. 7A and 7B.DiscussionSevere invasive S. agalactiae infections are not only a major cause of infections in newborns but also in nonpregnant adults. Bacterial invasion and disease pathogenesis is a complex process that is achieved through numerous virulence factors. The S. agalactiae bhemolysin is considered as one of the most important virulence factors in this context. Invasive S. agalactiae infections are almost exclusively caused by b-hemolytic strains. However, the role of the S. agalactiae b-hemolysin in the molecular interaction with host cells is not completely understood.Interestingly, the cov (or csr) system as a major regulator of S. agalactiae virulence genes suppresses b-hemolysin expression [19] [9]. A recent study by Sendi et al. found that a strain carrying a cov mutation resulting in a high hemolytic S. agalactiae variant with low capsule expression showed low intracellular survival in human neutrophils in contrast to the low hemolytic variant with high capsule expression [8]. To elucidate whether the observed difference is due to b-hemolysin or capsule expression, we used a serotype Ia strain and its isogenic nonhemolytic mutant to investigate the potential factors responsible for the different survival in eukaryotic host cells. The b-hemolytic S. agalactiae wild type strain was found in lower numbers in the intracellular compartment of THP-1 macrophages in comparison to the nonhemolytic mutant strain. With increasing incubation time (from 0.75 to 1.5 h), the number of reco.

Imental group, based on the absorbance at 450 nm of the reduced CCK-8 reagent, using an automicroplate reader (Flexstation 3, Molecular Devices, USA). Cell viability was expressed as the percentage of viable cells relative to the counts of untreated cells. Each experiment was conducted twice. Data were MedChemExpress SMER28 averaged and one representative experiment was shown.Results Changes in TGF-b1 and TGF-b2 Expression in Dysplasiacarcinoma SequencePositive TGF-b1 was present not only in dysplastic or malignant epithelial cells at the top of the gland, adjacent to the lumen, but also strongly in smooth muscle actin expressing fibroblasts (TA-02 biological activity Figure 1A?C). Positive staining for the 12926553 intracellular form of TGF-b1 occurred in 20 of the control samples, 52.3 of PC, 59.1 of EGC, and 66.7 of AGC samples. Linear tendency test showed that positive immunostaining rates for TGF-b1 were positively correlated with lesion progression (x2 = 9.487, P = 0.002). The histology of GC was then divided into `intestinal’ and `diffuse’ types, according to the criteria of Lauren [31]. Among the GC samples, 64.1 of intestinal-type showed strong immune reactivity and 63.6 of the diffuse-type were weakly stained. All tissues were stained positive for TGF-b2 (Figure 1D). There was no difference in the expression of TGF-b1 in relation to Helicobacter pylori (Hp) infection, Lauren’s classification or lymph node involvement (Table 1).Changes in TGF-b1 and TGF-b2 mRNA in Gastric PC and Cancer TissuesTGF-b1 mRNA levels were increased in AGC, while TGF-b2 levels were enhanced in EGC. TGF-b1 mRNA levels increased significantly from the control, PC, EGC, and AGC stages (Figure 2A; P,0.05). Sub-analysis demonstrated that TGF-b1 mRNA levels were significantly higher in AGC compared to the PC and control groups (P,0.05), while TGF-b2 levels were increased in EGC and AGC, compared to the control group (P,0.01) (Figure 2B). Furthermore, TGF-b1 mRNA levels were higher in tumor than in peritumor (P,0.001) (Figure 2C); however, TGF-b2 levels demonstrated the opposite tendency (P,0.05) (Figure 2D). In addition, correlation analysis identified positive correlations between the mRNA levels of TGF-b1 and Smad2 (r = 0.346, P = 0.025) and Smad7 (r = 0.461, P = 0.002) (Figure 2E and 2F). TGF-b2, however, showed no association with Smad2 or Smad7, and neither TGF-b1 nor TGF-b2 was correlated with Smad3 or Smad4. Taken together, these results suggest that TGF-b1 and TGF-b2 might play different roles in tumor progression.Enzyme-linked Immunosorbent Assay (ELISA)TGF-b1 and TGF-b2 levels in monoculture and coculture systems were determined by sandwich ELISA using Quantikine human TGF-b1 immunoassay and TGF-b2 immunoassays (R D Systems, USA), according to the manufacturer’s instructions. A total 15755315 of 100 mL of cell supernatants from the direct and indirect culture groups, respectively, were treated with 20 mL of 1 M Hcl for 10 min, followed by neutralization with 20 mL of 1.2 M NaOH. The samples were then pipetted into microplate wells precoated with a monoclonal antibody specific for TGF-b1, and incubated for 2 h at room temperature. An enzyme-linked polyclonal antibody specific for TGF-b1 was then added to the wells and incubated for a further 2 h to sandwich the TGF-b1 ligand. A substrate solution consisting of hydrogen peroxide and tetramethyl benzidine was added and the intensity of the color was determined using an automicroplate reader (Flexstation 3, Molecular Devices). Each experiment was conducted twi.Imental group, based on the absorbance at 450 nm of the reduced CCK-8 reagent, using an automicroplate reader (Flexstation 3, Molecular Devices, USA). Cell viability was expressed as the percentage of viable cells relative to the counts of untreated cells. Each experiment was conducted twice. Data were averaged and one representative experiment was shown.Results Changes in TGF-b1 and TGF-b2 Expression in Dysplasiacarcinoma SequencePositive TGF-b1 was present not only in dysplastic or malignant epithelial cells at the top of the gland, adjacent to the lumen, but also strongly in smooth muscle actin expressing fibroblasts (Figure 1A?C). Positive staining for the 12926553 intracellular form of TGF-b1 occurred in 20 of the control samples, 52.3 of PC, 59.1 of EGC, and 66.7 of AGC samples. Linear tendency test showed that positive immunostaining rates for TGF-b1 were positively correlated with lesion progression (x2 = 9.487, P = 0.002). The histology of GC was then divided into `intestinal’ and `diffuse’ types, according to the criteria of Lauren [31]. Among the GC samples, 64.1 of intestinal-type showed strong immune reactivity and 63.6 of the diffuse-type were weakly stained. All tissues were stained positive for TGF-b2 (Figure 1D). There was no difference in the expression of TGF-b1 in relation to Helicobacter pylori (Hp) infection, Lauren’s classification or lymph node involvement (Table 1).Changes in TGF-b1 and TGF-b2 mRNA in Gastric PC and Cancer TissuesTGF-b1 mRNA levels were increased in AGC, while TGF-b2 levels were enhanced in EGC. TGF-b1 mRNA levels increased significantly from the control, PC, EGC, and AGC stages (Figure 2A; P,0.05). Sub-analysis demonstrated that TGF-b1 mRNA levels were significantly higher in AGC compared to the PC and control groups (P,0.05), while TGF-b2 levels were increased in EGC and AGC, compared to the control group (P,0.01) (Figure 2B). Furthermore, TGF-b1 mRNA levels were higher in tumor than in peritumor (P,0.001) (Figure 2C); however, TGF-b2 levels demonstrated the opposite tendency (P,0.05) (Figure 2D). In addition, correlation analysis identified positive correlations between the mRNA levels of TGF-b1 and Smad2 (r = 0.346, P = 0.025) and Smad7 (r = 0.461, P = 0.002) (Figure 2E and 2F). TGF-b2, however, showed no association with Smad2 or Smad7, and neither TGF-b1 nor TGF-b2 was correlated with Smad3 or Smad4. Taken together, these results suggest that TGF-b1 and TGF-b2 might play different roles in tumor progression.Enzyme-linked Immunosorbent Assay (ELISA)TGF-b1 and TGF-b2 levels in monoculture and coculture systems were determined by sandwich ELISA using Quantikine human TGF-b1 immunoassay and TGF-b2 immunoassays (R D Systems, USA), according to the manufacturer’s instructions. A total 15755315 of 100 mL of cell supernatants from the direct and indirect culture groups, respectively, were treated with 20 mL of 1 M Hcl for 10 min, followed by neutralization with 20 mL of 1.2 M NaOH. The samples were then pipetted into microplate wells precoated with a monoclonal antibody specific for TGF-b1, and incubated for 2 h at room temperature. An enzyme-linked polyclonal antibody specific for TGF-b1 was then added to the wells and incubated for a further 2 h to sandwich the TGF-b1 ligand. A substrate solution consisting of hydrogen peroxide and tetramethyl benzidine was added and the intensity of the color was determined using an automicroplate reader (Flexstation 3, Molecular Devices). Each experiment was conducted twi.

Resistance by the forced-oscillation technique, mice were exposed to increasing doses of methacholine (0.1?0 mg/ml), each of which was administered over 10 seconds by nebulization. Methacholine responsiveness in unsensitized, uninfected BALB/c mice was similar to our previous study [18], and did not differ from alum-sensitized controls (data not shown). As in other studies [11,20,25,26], significant airway hyperresponsiveness to methacholine was present in OVA-sensitized, uninfected animals (Fig. 3A). In contrast, airway hyperresponsiveness to methacholine was absent in OVA-sensitized mice following infection with RSV at both 2 and 8 d.p.i. Indeed, methacholine responsiveness was no different from that of unsensitized, RSVinfected animals at these same Lecirelin timepoints. Importantly, methacholine hyperresponsiveness was present in mice “infected” for 2 or 8 days with RSV which was antigenically-intact but replicationincompetent as a result of exposure to UV light (Fig. 3B) [16]. This latter finding indicates that viral replication is necessary for reversal of methacholine hyperresponsiveness in OVA-sensitized mice, and that this phenomenon does not result solely from administration of large amounts of viral antigen [27]. Moreover, itRSV reverses AHR in OVA-Sensitized MiceFigure 2. RSV infection reduces bronchoalveolar lavage fluid cell counts in OVA-sensitized mice. Effect of OVA sensitization on (A) Alveolar macrophage (AM), eosinophil (EO) and small lymphocyte (SL) counts in uninfected mice (OVA/UNINF; n = 8) and unsensitized, uninfected controls (UNSENS/UNINF; n = 8); (B) Total cell counts in OVA-sensitized mice after mock infection for 2 days (M2; n = 5), 4 days (M4; n = 4), or 8 days (M8; n = 8), and infection with RSV (106 pfu/mouse) for 2 days (n = 16), 18055761 4 days (n = 10), or 8 days (n = 6); (C) AM, EO, and SL counts after mock infection for 2, 4, or 8 days and infection with RSV for 2? days; and (D) Neutrophil (PMN) counts after infection with RSV for 2? days. No PMNs were detected in bronchoalveolar lavage fluid from uninfected or mock-infected mice at any timepoint (not shown). *P,0.05, **P,0.005, ***P,0.001. N.D.: None detected. doi:10.1371/journal.pone.0046660.gdemonstrates that reversal of methacholine hyperresponsiveness following RSV infection cannot be attributed to any contaminating mediators from the HEp-2 cells in which the virus is cultured, since these would remain functional following UV-crosslinking of viral RNA [16,18,28].RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice via a pertussis toxin-sensitive pathway. McGraw et al. have reported thatunsensitized, uninfected buy LED 209 b2-adrenergic receptor-knockout FVB mice are hyporesponsive to methacholine [22,29]. They also showed that pertussis toxin enhanced bronchoconstriction to methacholine, indicating that hyporesponsiveness in their model is mediated by G protein-inhibitory a subunit (Gai). Similarly, we found that treatment of OVA-sensitized, RSV-infected mice with pertussis toxin (100 mg/kg in 100 ml saline, i.p.), but not the vehicle control (100 ml saline only, i.p.), 18 hours prior to lung function analysis increased responsiveness to methacholine at 2 d.p.i. (Fig. 4). However, pertussis toxin pretreatment was insufficient to induce the airway hyperresponsiveness observed in untreated, OVA-sensitized, uninfected mice.Keratinocyte cytokine released in response to RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mic.Resistance by the forced-oscillation technique, mice were exposed to increasing doses of methacholine (0.1?0 mg/ml), each of which was administered over 10 seconds by nebulization. Methacholine responsiveness in unsensitized, uninfected BALB/c mice was similar to our previous study [18], and did not differ from alum-sensitized controls (data not shown). As in other studies [11,20,25,26], significant airway hyperresponsiveness to methacholine was present in OVA-sensitized, uninfected animals (Fig. 3A). In contrast, airway hyperresponsiveness to methacholine was absent in OVA-sensitized mice following infection with RSV at both 2 and 8 d.p.i. Indeed, methacholine responsiveness was no different from that of unsensitized, RSVinfected animals at these same timepoints. Importantly, methacholine hyperresponsiveness was present in mice “infected” for 2 or 8 days with RSV which was antigenically-intact but replicationincompetent as a result of exposure to UV light (Fig. 3B) [16]. This latter finding indicates that viral replication is necessary for reversal of methacholine hyperresponsiveness in OVA-sensitized mice, and that this phenomenon does not result solely from administration of large amounts of viral antigen [27]. Moreover, itRSV reverses AHR in OVA-Sensitized MiceFigure 2. RSV infection reduces bronchoalveolar lavage fluid cell counts in OVA-sensitized mice. Effect of OVA sensitization on (A) Alveolar macrophage (AM), eosinophil (EO) and small lymphocyte (SL) counts in uninfected mice (OVA/UNINF; n = 8) and unsensitized, uninfected controls (UNSENS/UNINF; n = 8); (B) Total cell counts in OVA-sensitized mice after mock infection for 2 days (M2; n = 5), 4 days (M4; n = 4), or 8 days (M8; n = 8), and infection with RSV (106 pfu/mouse) for 2 days (n = 16), 18055761 4 days (n = 10), or 8 days (n = 6); (C) AM, EO, and SL counts after mock infection for 2, 4, or 8 days and infection with RSV for 2? days; and (D) Neutrophil (PMN) counts after infection with RSV for 2? days. No PMNs were detected in bronchoalveolar lavage fluid from uninfected or mock-infected mice at any timepoint (not shown). *P,0.05, **P,0.005, ***P,0.001. N.D.: None detected. doi:10.1371/journal.pone.0046660.gdemonstrates that reversal of methacholine hyperresponsiveness following RSV infection cannot be attributed to any contaminating mediators from the HEp-2 cells in which the virus is cultured, since these would remain functional following UV-crosslinking of viral RNA [16,18,28].RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice via a pertussis toxin-sensitive pathway. McGraw et al. have reported thatunsensitized, uninfected b2-adrenergic receptor-knockout FVB mice are hyporesponsive to methacholine [22,29]. They also showed that pertussis toxin enhanced bronchoconstriction to methacholine, indicating that hyporesponsiveness in their model is mediated by G protein-inhibitory a subunit (Gai). Similarly, we found that treatment of OVA-sensitized, RSV-infected mice with pertussis toxin (100 mg/kg in 100 ml saline, i.p.), but not the vehicle control (100 ml saline only, i.p.), 18 hours prior to lung function analysis increased responsiveness to methacholine at 2 d.p.i. (Fig. 4). However, pertussis toxin pretreatment was insufficient to induce the airway hyperresponsiveness observed in untreated, OVA-sensitized, uninfected mice.Keratinocyte cytokine released in response to RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mic.