Observations of that strain from Abu Dhabi [2] and in German patients with family ties to Turkey [14] as well as the present study might suggest

that this strain is common and widespread in the Middle East. PVL-positive CC30-IV is a strain mainly known from the Pacific islands, Samoa and New Zealand, but also from Abu Dhabi [2] and Kuwait [8]. An importation of that strain into Gulf countries appears to be likely due to the high numbers of immigrant labourers from Pacific countries such as the Philippines, as similarly noted in Denmark [36]. PVL-positive CC80-IV has been dubbed the European CA-MRSA strain as it is widespread although sporadically detected across several European countries. However, it appears to be more predominant in the Middle East and Maghreb (North African) countries being detected Ibrutinib chemical structure not only in Saudi Arabia but also in Abu Dhabi [2], Kuwait [37], Lebanon [9], Tunisia [11] and Algeria [12]. Other strains were rare being identified only in sporadic cases, accounting for less than 3% each. Some of the minor strains have been previously observed in other ERK inhibitor regions so that an importation might be likely. For others no, or only few, data on distribution or prevalence are available. Therefore it is not clear if they emerged locally or if they have been imported. For instance, CC1/ST772-V is known to mainly occur in India and Bangladesh, and cases in Europe

are usually linked to these countries [35, 38]. There might also be an epidemiological link to India for the isolate from this study, as there are high numbers of Indian workers, including healthcare workers, in Riyadh. CC5-IV is known to occur essentially worldwide. CC5-IV/SCCfus has been described only from Malta [22], so it would be interesting to check whether this strain has a wider distribution in the Mediterranean countries and the Middle East. CC6-IV has previously been observed not only in Australia, but also in Abu Dhabi [2]. Interestingly, CC6-MSSA has been found to be a common clone

in Middle Eastern camels [39] so that a local emergence of CC6-IV after inter-species transfer and acquisition of a SCCmec element appears to be possible. PVL-negative CC80-IV appear to be extremely scarce, and the few detected isolates might be deletion variants of the so-called European CA-MRSA clone. One of the two isolates identified in this study carried enterotoxin genes, FER which is also a rare feature among CC80. PVL-positive CC88-IV are known from Abu Dhabi and, sporadically, from Europe. CC97-V has been previously identified in Egypt, which warrants further study on its presence in the Middle East. Since CC97 MSSA are common among domestic animals, here again a possible transmission from livestock should be investigated. The MRSA strains found in Saudi Arabian patients showed a significantly high carriage of PVL genes (54.21%). Comparable high figures have been reported from Algeria [13] as well as from Abu Dhabi (41.9%, [2]).

Methods in enzymology: Academis Press Inc; 1994. 73. Taguchi F, Ogawa Y, Takeuchi K, Suzuki T, Toyoda K, Shiraishi T, Ichinose Y: A homologue of the 3 oxoacyl- (acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates

virulence factors via N-acyl homoserine lactona and fatty acid synthesis. J Bacteriol 2006, 188:8376–8384.PubMedCrossRef Competing interests The authors have declared that no competing interests exist. Authors’ contributions JLA-G and AH-M contributed to experimental design, performed experiments, analyzed data, and drafted the manuscript. JRP-A performed experiments and analyzed data. AA-M conceived the study, contributed to experimental design, and edited the manuscript. All authors read and approved the final manuscript.”
“Background

Members of Vibrionaceae (Gammaproteobacteria: NVP-LDE225 concentration Vibrionales) have been known since 1854 (Pacini) and were shown to be distinct much before pulsed-field gel electrophoresis this website revealed the most distinct diagnostic “morphological” feature, the existence of two chromosomes [1]. The interest in these bacteria is not surprising given that several species are pathogenic to humans and marine organisms and others are bioluminescent symbionts of marine fishes and squids e.g.[2–7]. Some lesser-known species are psychrophiles (live in cold temperatures), piezophiles (live at high pressures), or halophiles (live at high NaCl concentration; [8]). The diversity of ecologies represented by members of Vibrionaceae has led to enthusiastic genome sequencing in the group, which has focused most heavily

on pathogenic species (more than 31 strains of V. cholerae are available on GenBank as of 2012). A phylogenetic hypothesis based on complete genomes was desired for Vibrionaceae. While the analysis presented in [9] for Vibrionaceae was the most comprehensive to date (eight gene U0126 loci for 95 Vibrionaceae species) and provided the a hypothesis for a phylogenetic taxonomy for the group, the number of genomes already sequenced for Vibrionaceae lends itself to a genome-level analysis. While the specter of horizontal gene transfer always looms over phylogenetic analyses of bacteria, genome-level analyses take a proactive stance in the hopes of recognizing and quantifying problematic data partitions without blind dismissal of all phylogenetic signal. Because members of Vibrionaceae have two chromosomes, as discussed below, the genome-level phylogenetic analyses presented here provide phylogenetic evidence for the evolutionary scenarios that have been postulated for the maintenance of these two separate chromosomes. There are also many Vibrionaceae species that are present on GenBank as multiple contigs. This was not the case for members of Shewanellaceae, the sister taxon to Vibrionaceae, for which a genome-level phylogenetic hypothesis was presented in [10].

The MS/MS data were then

searched against a database indexed for only Clostridium spp. for protein identification. Whole genome sequencing and analysis Genomic DNA was isolated from strain CDC66177 using the MasterPure kit (Epicenter, Madison, WI) with modifications previously described [23]. This DNA was further purified using a Genomic-tip 100/G column (Qiagen, Valencia, CA). One microgram of genomic DNA was sheared using a Covaris S2 ultrasonicator system to a mean size of 1 Kb. The sheared DNA was used to construct a SMRTbell sequencing library (Pacific Biosciences) according to manufacturer’s instructions. The SMRTbell library was then bound into SMRTbell-DNA polymerase complexes and loaded into zero-mode waveguides (ZMW) on 4 SMRTcells selleck compound and sequenced using Pacific Biosciences C2 chemistry. This relatively small insert sized library was utilized to promote production of circular concensus reads (CCS) which retain higher accuracy

base calls than the longer continuous length reads (CLR). Eight 45 min movies were recorded and processed, yielding ~305 K reads with a mean readlength of 2.9 Kbases and total of Cabozantinib clinical trial 889 Mbases of sequence. CCS reads (140 K reads) were then used to error correct the longer (165 K reads) CLR reads [24] utilizing the Pacific Biosciences analysis script BLASR and then the combined CCS/corrected CLR fastq format reads were imported into CLC Genomics workbench. Sequence reads were then trimmed of any remaining Pacific Biosciences hairpin adaptor sequences and quality trimmed to a base Q value of 20. The filtered reads were then assembled de novo using the CLC denovo assembler. The 188,898 input reads provided a draft assembly of a 3.85 Mb genome comprised of 119 contigs with an N50 value of 87,742 bases with an average coverage of 28X. Annotation of the whole genome sequence was performed using RAST [25]. Pairwise alignments of various genes were made with EMBOSS Needle (http://​www.​ebi.​ac.​uk/​Tools/​psa/​emboss_​needle/​nucleotide.​html). ANI values were determined

using the computer program JSpecies [17]. MLST loci from selected previously reported type E strains were obtained from Genbank [11]. These MLST loci were used to search for the corresponding alleles in the strain 17B genome sequence and enough the CDC66177 whole genome sequence using BLAST. Concatemers of the alleles for each strain were generated and a multiple sequence alignment was performed using CLUSTALW because the lengths of some alleles in strains 17B and CDC66177 differed due to insertion and/or deletions. Acknowledgements Sanger sequencing was performed in the Genomics Unit within the Division of High Consequence Pathogens and Pathology at CDC. This publication was supported by funds made available from the Centers for Disease Control and Prevention, Office of Public Health Preparedness and Response.

In addition, other than the report by Kramer et al. [30], with its noted limitations, no population-level data reported on the epidemiology of PASS

across the full spectrum of pregnancy outcomes, including induced abortion, miscarriage, antepartum and postpartum hospitalizations. Only one study to date Wnt inhibition has described trends of the incidence of PASS. Bauer et al. [33] reported that the incidence of PASS rose 10% per year between 1998 and 2008. The incidence of PASS increased from 7 to 14 hospitalizations per 100,000 deliveries over study period. However, the sources of rising incidence of PASS remain unclear. Several investigators have noted the rising incidence of conditions and procedures leading to maternal severe sepsis and septic shock, including rising maternal age, obesity, chronic illness, use of cesarean section, and use of invasive procedures [25]. While the aforementioned factors are well associated with risk of infection,

their role in progression from infection to severe sepsis among obstetric patients has not been systematically examined. Indeed, the changes in the frequency of the aforementioned risk factors over time among the patients reported by Bauer et al. [33] have not been reported and require further study. Only a few studies on the relative development of PASS across different phases of pregnancy have been reported and varied markedly across cohorts. selleck inhibitor PASS related to abortion was reported in 6% [27] to 7% [35]. Development of PASS during the antepartum period occurred between 33% [30] and 73% [35], while postpartum PASS events were noted to account for 20% [35] and up to 92.9% [29] of all PASS events. The marked differences in the relative occurrence of PASS across different phases and outcomes of pregnancy reported in the aforementioned studies likely reflects unique local population characteristics, selection bias, and the small Acetophenone sample size. Further larger population-level studies are needed to better understand the risk of PASS across

non-delivery phases of pregnancy. The demographic characteristics of women developing PASS varied with the studied populations. The average age reported ranged from 25.8 years [27] to 32 years [30]. The rate of PASS event in teens and among women older than 34 years was described infrequently, reported in 13.6% and 19.9%, respectively [33]. Black women constituted between 7.1% [29] and 56% [27] of PASS cohorts in local studies and between about 9% [32] and 21.2% [33] in population-level reports, while Hispanic women were reported in 13% [35] and 56.4% [32] of PASS events, reflecting regional variations. Health insurance among US patients with PASS has been reported in two studies. Medicaid was the predominant health insurance (49.8%) of women nationally in the study by Bauer et al. [33], with 3.6% lacking health insurance. Acosta et al. [32] reported the combination of public health insurance/no insurance in 58.2% of PASS hospitalizations.

The experiment was repeated twice. To validate the interaction data by an independent approach, we selected some of the VipA mutants and tested them for binding to VipB in the Y2H system using two independent reporter genes: lacZ, which allows us to compare the relative strength of the VipA-VipB interactions by quantification of β-galactosidase activity, and MEL1, which in the case of a positive interaction and in the presence of the substrate X-α-Gal will promote blue color development. According to both reporters, the deletion mutant Δ104-113, the double

mutant V110A/L113A and the quadruple mutant D104A/V106A/V110A/L113A were all essentially unable to bind VipB and produced α-and β-galactosidase levels similar to the negative vector control, while the double mutant D104A/V106A and the triple mutant D104A/V106A/V110A

both showed intermediate binding (Table 1 and data not shown). The less sensitive MEL1 reporter click here assay did not detect any obvious binding defects for single mutants D104A, V106A or V110A (data not shown), while the lacZ reporter revealed a weak binding defect for both V106A and V110A mutants (Table 1). Thus, overall, the Y2H data confirms the results from the E. coli B2H assay. Table 1 Protein-protein interactions in the yeast two-hybrid assay DNA-binding domain Activation domain Relative β-gal activity VipB None 0.5 ± 0.1% *** VipB VipA 100.0 ± 5.8% VipB VipA Δ104-113 1.0 ± 0.2% *** VipB VipA D104A 92.7 ± 4.1% VipB VipA V106A 92.4 MLN0128 price ± 3.4% * VipB

VipA V110A 74.6 ± 3.4% *** VipB VipA D104A/V106A 64.1 ± 10.7% * VipB VipA V110A/L113A 1.1 ± 0.3% *** VipB VipA D104A/V106A/V110A 48.8 ± 2.0% *** VipB VipA D104A/V106A/V110A/L113A 1.0 ± 0.2% *** VipA mutants fused to the GAL4 activation domain of plasmid pGADT7 were co-transformed with VipB on the GAL4 DNA-binding domain pGBKT7 into the S. cerevisiae reporter strain Y187. Activation of the lacZ reporter from 4 independent experiments where duplicate transformants were tested on each occasion was determined and expressed as % mean β-galactosidase activity ± SEM relative to the activity of the wild-type protein. A Student’s 2-sided t-test was used to determine whether the differences observed were statistically significant (*, P < 0.05; ***, P < 0.001). Recently, we have shown that temperature and salinity influences the activity of the T6SS of V. cholerae O1 strain Adenosine A1552 [13]. To determine whether salt and/or temperature also influence(s) the interaction of VipA and VipB, we compared the strength of the interaction in the B2H assay when E. coli was grown under different salt and temperature conditions. The results suggest that E. coli grown in Luria Broth (LB) supplemented with additional NaCl (high salt) over night, generally produce higher β-galactosidase activity than if grown in low salt (i.e. normal LB) (Figure 3). This suggests that a high concentration of salt is beneficial for the VipA-VipB interaction.

Sections were slightly counterstained with Mayer’s hematoxylin and mounted in aqueous mounting medium (Glicergel, Dako). Dako control slides were used as positive controls and the negative control was performed by omitting the application of the primary antibody. IHC scoring was based on the membrane immunoreactivity, according to the American Joint

Committee [17]: 0, no reactivity, 1+, weak reactivity, 2+, moderate reactivity, 3+, strong reactivity. Chromogenic in situ hybridization Formalin fixed paraffin embedded (FFPE) sections were deparaffinized, dehydrated, learn more air dried, and heated in boiling tissue heat pre-treatment buffer for 15 minutes using a SPoT-Light® FFPE reagent kit (Zymed, Histoline, Milan, Italy). Enzymatic digestion was performed using SPoT-Light® FFPE digestion enzyme (Zymed) for 2-3 minutes at RT. After dehydration, histological slides were air dried and the ready-to-use double-stranded DNA digoxygenin-labelled EGFR probe (Zymed) or the biotin labelled chromosome 7 centromeric probe (Zymed) were applied. Denaturation was performed by incubating the slides, covered with a CISH cover-slip, on a 96°C heating block for 5 minutes, and hybridization was performed by placing the slides in a humidity chamber at 37°C overnight. After removing the cover-slips, a stringent wash was performed in 0.5× saline-sodium citrate buffer

at 80°C for 5 minutes. The endogenous peroxidase activity and unspecific staining were blocked https://www.selleckchem.com/products/Methazolastone.html by applying 3% hydrogen peroxide and the CAS-Block™, respectively.

A mouse antidigoxygenin antibody was added to the slides hybridized with EGFR probe for 45 minutes at RT followed by incubation with a polymerized peroxidase-goat anti-mouse antibody (Dako) for 45 minutes at RT. On the FFPE tissue slides, the colorimetric signal of chromosome 7 centromeric probe was improved by incubating mafosfamide the slides with a mouse antibiotin antibody (Dako) for 45 minutes at 37°C. A DAB chromogen substrate system was used to generate a sensitive signal that could be viewed with a Nikon ECLIPSE 55i transmission light-brightfield microscope (Nikon, Amstelveen, The Netherlands) after Mayer’s haematoxylin counterstaining. Fluorescence in situ hybridization FISH was performed using the LSI EGFR (SpectrumOrange™), a locus-specific probe for the EGFR human gene locus (7q12) and the chromosome enumeration probe (CEP 7, SpectrumGreen™) for alpha-satellite DNA located at the centromere (7q11.1-q11.1) (Vysis, Inc., Downers Grove, IL). The assay was carried out according to the manufacturer’s instructions. Shortly after deparaffinization, the FFPE specimens were incubated in the pre-treatment solution (82°C, 30 minutes) and then digested with protease (37°C, 15 minutes). After washing, the slides were counterstained with 4′,6-diamidino-2 phenylindole (DAPI) and analyzed using a fluorescent microscope. An average of 30 nuclei was counted for each case.

Clinicians believed that they were the most appropriate group, while geneticists and experts with a bioethical background thought that results should be disclosed by a multidisciplinary team. This team should consist of not only clinicians but also other professionals, such as geneticists and clinicians specialised in the relevant condition (e.g. oncologist if a cancer susceptibility gene had been discovered).

At the same time, most of the experts Cell Cycle inhibitor questioned the appropriateness of clinicians not specialised in genetics dealing with genetic tests and the results, especially when NGS is used. They were of the opinion that non-specialist clinicians lacked the expertise to explain the procedures and to provide pre- and post-testing counselling. The lack of a recognised specialty of “clinical geneticist” made things even harder. To understand that, here we are acting as genetic counsellors because compound screening assay we don’t have genetic counsellors and doctors don’t know what to do. They are asking for our help and sometimes even we don’t know what to do (Participant 05). Not to mention that we don’t even have a specialty recognised! (Participant 02) Which results should be returned? Most experts mentioned the concept

of “patient autonomy” and understood this as each patient’s individual right to choose whether or not to be told about IFs, although their ideas about the best way to achieve this varied. We need to make sure that they are informed well enough and that they are deciding autonomously.

We should give them all the information we can and let them decide by themselves (Participant 03). Whoever is doing the genetic counselling should provide all the available information. They should let them know that IFs could be discovered. And then it is on the individual’s responsibility these to ask his doctor if they indeed discovered something. This way we would be sure that the individual actually wants to learn the findings. If it is the doctor that asks then that is not exactly autonomous! They need to actively participate! (Participant 01) However, it seems current practice is not always guided by this principle. Clinicians admitted they do occasionally adopt a more paternalistic approach and try to act in what they think is their patient’s best interest, even if this means making some preliminary decisions by themselves. Even if the patient has asked for all results we won’t give him everything. We will definitely give him clinically valid and clinically actionable ones, or results that concern serious of life-threatening conditions but about the rest of them … I don’t know. We will discuss about it and according to what we will decide we will let him know (Participant 06). We won’t give him everything. We will discuss it and we will decide what he needs to know (Participant 08).

Clinicians believed that using NGS in the clinical setting would create problems because “if you start looking, you will definitely find something”. Therefore, for the time being, targeted sequencing would be more useful. For me it is rather simple. If symptoms resemble Huntington’s for example Ibrutinib concentration I will order a test only for that. I won’t start looking around. I won’t even use genetic testing unless I have to. I am not saying that it is not useful, because it is, and occasionally we have managed to diagnose conditions

that we couldn’t have done otherwise, but if I can use other kinds of testing I would rather do that. With genetic testing you never know what you will get (Participant 10). Not even for cancer. If later we discover that all cancers are hereditary maybe then but until then I would only use genomic testing rarely in extreme cases (Participant 04). Although Greek experts noted selleck chemicals llc that there are some similarities with other areas of medical practice that can provide a starting point, clinicians

reported that the concept of IFs is well integrated in the medical philosophy and they have been “taught” how to handle them during their medical training. But IFs are not something you could only have in genetic testing. We always knew that could happen (Participant 04). Most tests could give you IFs. We have been trained and we always knew that the more you look the more you will find. It might be even more with genetic testing but the idea is the same (Participant 10). Additionally, they all reported having experience of handling IFs from other types of genetic testing and thought this would be of some help when dealing with IFs deriving from NGS testing. We have been thinking about this for a long time now. Especially with arrays [array-CGH (Comparative Genomic

Hybridization)] we have found unexpected things more than FER once. It’s not something new (Participant 05). Oh, yes. We are used to having IFs. We have them in prenatal testing very often. Ever since we started using the classical karyotype. You are looking for one thing and you find something else. Now we are going to use all this experience for clinical sequencing. This is not new to us (Participant 07). Previous experience from other types of testing could inform practices about IFs from clinical sequencing (e.g. IFs discovered during prenatal tests using cytogenetic tests); yet, experts considered that IFs differ in important ways. First, all participants reported that a very important difference was that genetic information affects more than just the actual patient or the person getting tested. The nature of genetic information makes it unique and complex because it is shared by all family members, even those not affected by the genetic condition in question. What is different this time is that family members have even a legal right to have access to that information.

Ann For Sci 65:309CrossRef Foody GM, Jackson RG, Quine CP (2003) Potential improvements in the characterisation of forest canopy gaps caused by windthrow using fine resolution multispectral data: comparing hard and HM781-36B soft classification techniques. For Sci 49:444–454 Forster B (1998) Storm damages and bark beetle management: how to set priorities. In: Grodzki W, Knížek M, Forster B (eds) Methodology of forest insect and disease survey in Central Europe. IUFRO—Forest Research Institute, Warsaw, pp 161–165 Gibb H,

Hjältén J, Atlegrim O, Hilszczański J, Ball JP, Johansson T, Danell K (2006a) Effects of landscape composition and substrate availability on saproxylic beetles in boreal forests: a study using experimental logs for monitoring assemblages. Ecography 29:1–14CrossRef Gibb H, Pettersson Cisplatin RB, Hjältén J, Hilszczański J, Ball JP, Johansson T, Atlegrim O, Danell K (2006b) Conservation-oriented forestry and early successional saproxylic beetles: responses of functional groups to manipulated dead wood substrates. Biol Conserv 129:437–450CrossRef Gilbert M, Nageleisen LM, Franklin A, Grégoire JC (2005) Post-storm surveys reveal large-scale spatial patterns and influences of site factors, forest structure and diversity in endemic bark-beetle populations. Landsc

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much (L.) (Col.: Scolytidae) to changing breeding in a forest decline area in the Sudeten Mountains, Poland. J Pest Sci 77:43–48CrossRef Grodzki W (2007) Wykorzystanie pułapek feromonowych do monitoringu populacji kornika drukarza w wybranych parkach narodowych w Karpatach. Pr IBL, Rozpr Monogr 8:1–128 Grodzki W, Loch J, Armatys P (2006a) Występowanie kornika drukarza Ips typographus L. w uszkodzonych przez wiatr drzewostanach świerkowych masywu Kudłonia w Gorczańskim Parku Narodowym. Ochr Besk Zach 1:125–137 Grodzki W, Jakuš R, Lajzová E, Sitková Z, Mączka T, Škvarenina J (2006b) Effects of intensive versus no management strategies during an outbreak of the bark beetle Ips typographus (L.) (Col.: Curculionidae, Scolytinae) in the Tatra Mts. in Poland and Slovakia. Ann For Sci 63:55–61CrossRef Grodzki W, Kosibowicz M, Mączka T (2008) Skuteczność wystawiania pułapek feromonowych na kornika drukarza Ips typographus (L.) w sąsiedztwie wiatrowałów i wiatrołomów. Leś Pr Bad 69:365–370 Grodzki W, Turčáni M, Jakuš R, Hlásny T, Raši R, McManus ML (2010) Bark beetles in the Tatra Mountains. International research 1998–2005—an overview. Fol For Pol Ser A 52:114–130 Haase P (1995) Spatial pattern in ecology based on Ripley’s K-function: introduction and methods of edge correction.

Methods HBV Plasmids Five HBV 1.35-fold genome plasmids – N10 (genotype Ae, AY707087), C4371 (genotype Ba, GU357842), Y1021 (genotype C1, GU357845), Y10 (genotype D1, GU357846) and W29 (genotype I1, GU357844) were used for transfection and hydrodynamic injection. The constructions and molecular

and phenotypic characteristics are described in our previous report [36]. Bioinformatics Analysis To define the conservative sites on HBV genomes amongst the various genotypes, all available complete genome sequences selleck of HBV, as of April 2009, were downloaded from GenBank. Multiple alignment was done with ClustalX2 under default settings (Gap Opening:10, Gap Extension: 0.2, Delay Divergent Sequences(%): 30, DNA Transition Weight: 0.5, Use Negative Matrix: Off). The most representative and informative sequence in terms of phylogeny were collected as a dataset and the most similar sequences were removed using all

pairwise distance scan. A total of 327 HBV genomes including A-I genotypes and nearly all reported subtypes were remained in the final dataset. The genotypes and subtypes of six HBV genomes isolated in the study were submitted to phylogenetic analysis using MEGA 4.0 software (data not shown). Forty sites with conservative sequences were selected and the shRNA plasmids were constructed (Table 1). The designed siRNA were evaluated for potential off-target effects by the online SOS program http://​rnai.​cs.​unm.​edu/​offTarget. The sequences and positions Wnt beta-catenin pathway of the forty designed shRNA targets are shown in Table 1. ShRNA Plasmids ShRNA plasmids were cloned downstream of the human H1 promoter in the vector pSUPER [37]. The target sites for siRNA were

chosen based Y-27632 datasheet on conservative sites among the major HBV genotypes and subtypes. An shRNA plasmid targeting the firefly luciferase gene was used as a control (L1254: TGG CTA CAT TCT GGA GAC ATA). Cell Culture and In Vitro Transfection The plasmids used for in vitro transfection were purified with PlasmidSelect Xtra Starter Kit (GE Health, Sweden) and the concentrations were determined by the UV-spectrophotometric method. To determine the ability of siRNA to inhibit HBV gene expression in cell cultures, Huh7 cells were co-transfected with 4 μg of HBV plasmids, 1 μg of shRNA plasmids and 0.4 μg of a pcDNA3.1-SEAP plasmid using Lipofectamine 2000 (Invitrogen, Shanghai, China) following the manufacturer’s instructions. They were then harvested four days later. The pcDNA3.1-SEAP plasmid is a reporter plasmid expressing secreted alkaline phosphatase and used for transfection efficiency standardization by estimating SEAP enzymatic activity (Pierce; Kunming, China) in the culture supernatant.