Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in  General inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar YX4Lϕ motifs, and thus can be considered as molecular mimics of eIF4G, which compete for the same binding site of eIF4E, blocking the formation of the translation initiation complex, when hypophosphorylated in quiescent cells  to the mnt cells .Xenopus oocytes, 4E-T is a component of the large 2–3 MDa CPEB RNP (ribonucleoprotein) translational repressor complex  protein and its fly and worm homologues Cup and Spn-2 (Pqn-45 or IFET-1) that act in early development. In  complex . Cup and complex . Cup is  complex . Spn-2 l complex , ribonuc complex , and fun complex . As with complex ,19. Curr4Lϕ sequence that mediates eIF4E binding, and its nuclear export/import via Crm1 (Human 4E-T (h4E-T) was first characterized as a nucleocytoplasmic shuttling protein with a canonical YXvia Crm1 . With thvia Crm1 . Ectopicvia Crm1 ,22. Howevia Crm1 ,22, altoP-bodies, which contain mRNA, microRNAs, RNA-binding proteins/translational repressors and mRNA decay enzymes but not ribosomes, are thought to be involved in reversible translational repression including that mediated by microRNAs and in mRNA decay [reviewed by ]. To datThe molecular details of the P-body pathway in gene expression control remain to be determined, and available evidence does not offer a simple functional conclusion. Individual components—for example, GW182 and rck/p54—have well-characterized important functions in general and in miRNP-mediated gene expression control. However, the visible presence of P-bodies is not required for reporter mRNA decay mediated by microRNAs, siRNAs or via the NMD pathway, nor for general mRNA decay/translational repression in yeast . On the Drosophila (both Cup and the relatively uncharacterized CG32016 protein closer to human 4E-T than Cup) and Caenorhabditis elegans, and showed that conserved C-terminal sequences targeted 4E-T to P-bodies. In tether function assays, we showed that 4E-T represses translation of reporter mRNA in a P-body- and eIF4E-independent manner, and does not promote deadenylation. 4E-T mutated in its eIF4E-binding site modestly enhanced the decay of tethered mRNA, compared with wild-type 4E-T, due to increased binding of CNOT1/7 deadenylase subunits, likely accompanied by decapping. We also addressed the question of the cellular targets of 4E-T, and provide evidence that depleting 4E-T from HeLa cells significantly increased steady-state translation, in part at least due to partial relief of microRNA-mediated silencing.We were interested to investigate the role of P-bodies and eIF4E-binding in translational control mediated by 4E-T in mammalian cells, and to this end, we first delineated the sequences that target 4E-T to P-bodies. The deletion analysis of GFP-4E-T was based on comparison of sequences of vertebrate 4E-T proteins with their homologues in Drosophila Cup . While in general, the pattern of localization was similar for the constructs in both vectors, EGFP-C1 vectors showed a higher level of transfection and hence were used in all reported experiments. For the tethering constructs, the ORFs were first subcloned into pCI-HA or pCI-λNHA  and Renilla luciferase  with and without BoxB hairpins were described previously  with 10% fetal bovine serum. Cells were plated on a 20-mm coverslip in 35-mm plates (Thermo Scientific). Transfection was performed 24 h after the cells were seeded, using 4 µl of Lipofectamine 2000 (Invitrogen) and 2 µg of DNA constructs. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized using 0.5% Triton X-100 (Sigma) and then incubated with primary antibodies followed by staining with the appropriate Rhodamine-Red-X-conjugated (1:1000) (Jackson ImmunoResearch) secondary antibodies. Primary antibodies and dilutions were used as follows: 4E-T (1:200) (Abcam), eIF4E (1:1000) (Santa Cruz), rck/p54 (1:1000) (Bethyl Laboratories). To visualize the nucleus, cells were also stained with DAPI (1.25 µg/ml) for 10 s. Leptomycin B (in methanol) (Sigma) was used at a final concentration of 5 ng/ml for 5 h. Simultaneously, the same volume of methanol was added to the control cells. Coverslips were mounted with CitiFluor for visualization under the fluorescent Zeiss AxioImager M1 microscope or Leica SP5 confocal microscope. Each sample was analysed in triplicate. At least 100 transfected cells were analysed per sample.Saccharomyces cerevisiae AH109 cells as follows: 5 µl of salmon testes ssDNA (10 mg/ml), previously heated to 95°C for 10–20 min and cooled on ice, 100 µl of One-step-buffer , one yeast colony of ∼2 mm diameter and 0.5 µl of plasmid DNA (50–250 ng) were mixed by vortexing and incubated at 45°C, 30 min before plating on selective medium . Plates were incubated at 30°C for 3–5 days until colonies were 2–3 mm in diameter. A single colony was selected and re-streaked onto a fresh plate. These singly transformed strains were used as a yeast stock in which to transform the pGAD-protein constructs. The second round of transformation was performed in exactly the same way as described above with the exception that selection required –Trp-Leu dropout medium to select for both plasmids. Stocks were made from single colonies of double transformants and re-streaked on both –Trp-Leu and –Trp-Leu-Ade-His dropout plates to screen for plasmid retention and high-stringency protein interactions, respectively.pGBK-protein constructs were transformed into GFP-Trap® Magnetic beads (Chromotek) (25 µl) were resuspended in 250 µl of ice-cold NET buffer  supplemented with complete proteinase inhibitor cocktail (Roche) in hydrophobic tubes. Beads were washed by magnetic separation and lysates added (1000 µg in 1 ml NET buffer). Mixtures were incubated for 1.25 h at 4°C with constant rotation. Supernatants were removed after magnetic separation of samples, beads washed and protein eluted in 30 µl 1× SB. Fifteen microliters of input (40 µg) and output samples were loaded onto 15% SDS-PAGE or gradient gels and assessed by western blotting.Our standard 15% SDS-PAGE and western blotting procedures have been described previously . On occaRenilla luciferase reporter mRNA with 5 boxB sites and 100 ng of control firefly luciferase reporter mRNA. When DNA amounts between constructs were varied , they were substituted by respective amounts of pcDNA3 plasmid to make up 1 µg of DNA. After 48 h, cells were harvested and split between two tubes and used for either protein or RNA extraction. Protein extraction was performed using 1× Passive Lysis Buffer (Promega) on a rocker for 15 min at room temperature. Each sample was analysed in triplicate. The luciferase assay was performed as described (HEK293 cells were grown in DMEM+GlutaMAX (Gibco) with 10% fetal bovine serum. Cells were plated on 35-mm plates (Thermo Scientific). Transfection was performed 24 h after the cells were seeded, using 3 µl of Lipofectamine 2000 (Invitrogen) and 1 µg of DNA constructs encoding GFP, Pat1b, 4E-T WT and mutants fused to either HA-tag (control) or λNHA, 5 ng of escribed , using tSupplementary Table S1 and AMV reverse transcriptase (Promega). Quantitative RT-PCR reactions were performed using SYBR Green I (Sigma) mix in a Rotor-Gene 6000 (Corbett research) with the following cycling conditions: 95°C, 10 min; 40 × . To confirm specificity, amplification was followed by melting temperature analysis and agarose gel electrophoresis. In addition, minus reverse transcriptase controls were included to rule out contamination by genomic DNA. The relative change in mRNA levels was analysed using Rotor-Gene 6000 software package (Corbett Research/Qiagen). The fold change in mRNA levels was calculated relative to the control and normalized to GAPDH mRNA in triplicates in three independent experiments.RNA was extracted with Trizol (Invitrogen) following the manufacturer’s instructions and treated two times with RQ1 DNase I (Promega). RNA concentration was quantified using Nanodrop (Nanodrop Technologies). Two micrograms of RNA were converted to cDNA using primers specified in 32P] (Perkin Elmer). Probes corresponded to the full-length open reading frames, obtained by restriction digests and gel extraction (Qiagen kit). Hybridization of the probe was performed overnight in 5× SSC, 5× Dedhardt’s, 0.5% SDS, 0.1% sodium pyrophosphate, 100 µg/ml heparin solution at 65°C. The blot was washed 2 × 20 min at 60°C. Quantification was performed using Molecular Dynamics Storm 840 and ImageQuant software. Individual bands were measured and the background  was substracted. GAPDH RNA was used as a loading control.RNA was extracted using the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. RNA was denatured with glyoxal before electrophoresis on a 1.1% agarose gel and blotted overnight on to Hybond-N (Amersham) according to manufacturer’s instructions. The labelling probe was prepared using Amersham megaprime DNA Labelling System  and deoxycytidine 5-triphosphate,  and LSm1 were obtained from Life Technologies, and TNRC6A/B siRNAs were obtained from Life Technologies . HeLa cells were grown in DMEM with 10% FBS, with 30 000 cells seeded per well in 24-well plate format. siRNA (30 nM) was transfected with Dharmafect 1 transfection reagent (Thermo Fisher) as per manufacturer’s instruction. Subsequent transfection of luciferase reporter plasmids were carried out using Lipofectamine 2000 (Life Technologies) 48 h after siRNA transfections were performed as described . After a35S methionine/cysteine  for 45 min. Medium was removed after incubation and cells were washed twice with ice cold phosphate buffered saline before lysed with Passive Lysis Buffer (Promega). To determine the incorporation of 35S methionine isotopes, cell lysates were then precipitated with trichloroacetic acid precipitation on Whatman filter paper, while the protein content of each sample was determined with the Bradford protein assay (Bio-Rad). Data were collected from three independent experiments; error bars represent pooled standard deviation of each biological repeat.To determine the rate of new protein synthesis in control and 4E-T knock-down cells, HeLa cells were transfected with siRNA as described previously for 72 h. The final cell confluency did not exceed 80% in all cases. Medium was washed twice with phosphate buffered saline and incubated with methionine-free DMEM (Sigma-Aldrich) supplemented with 30 μCi/ml Danio rerio, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Bos taurus, Rattus rattus, Mus musculus, Homo sapiens and Gallus gallus) using ClustalW and Emboss Needle programs. Alignments between the vertebrate 4E-T proteins revealed a high degree of conservation throughout their sequences , which did not allow us to focus on any particularly conserved regions.In human tissue culture cells, 4E-T localizes to P-bodies and is required for P-body integrity ,21,43. TDrosophila melanogaster Cup, which is related to h4E-T in sequence, that regulates translation by interacting with 3′ UTR-binding proteins and eIF4E, and co-localizes with Me31B (rck/p54) in cytoplasmic particles in nurse cells (see ‘Introduction’ section). However, interestingly, despite their apparent common role in translational control, they do not share high sequence homology ; except for the canonical eIF4E-binding site and a region located towards the N-terminus of h4E-T and more centrally in Cup, which we named the Cup Homology Domain (CHD) . In addition, both proteins have C-terminal stretches of Q residues, particularly notable in Cup.Therefore, we expanded the analysis by including To further investigate the degree of functional conservation between human 4E-T and Cup, we compared their subcellular localization when tagged with GFP in human tissue culture cells. To this end, the full length Cup cDNA sequence was cloned into the EGFP-C1 vector and protein localization was examined using immunofluorescence microscopy. Cells were immunostained with rck/p54 antibodies as a marker for endogenous P-bodies. In contrast to h4E-T-GFP, which localized to P-bodies in >90% of cells, as well as being free in the cytoplasm mirroring the endogenous protein distribution, Cup-GFP was found diffused in the cytoplasm with weak nuclear localization, but showing no enrichment in P-bodies B.Drosophila Cup did not behave like 4E-T in HeLa cells was to an extent surprising, since other related proteins such as Pat1b and HPat localize to P-bodies in HeLa and S2 cells, respectively . Strikingly, unlike Cup, Dm4E-T-GFP showed P-body localization in the majority of transfected HeLa cells . Phylogenetic analysis confirmed that Dm4E-T (CG32016) and C. elegans Spn-2 , domain predictions (using DOMAC) and prediction of order and disorder regions (using POODLE). The protein sequence was truncated before or after the conserved regions identified in this search .To identify the regions of human 4E-T that mediate its P-body localization, we examined the cellular localization of 4E-T-GFP protein variants in HeLa cells, truncated from either the N- or the C-terminus. To define where to truncate 4E-T, we used the multiple sequence alignment of human, We initially tested C-terminally truncated proteins, with an intact eIF4E-binding site. The mutants truncated downstream of amino acids 845 and 716 localized to P-bodies as well as the full-length protein A. In conDm4E-T and CeSpn2, but absent from Cup, is important for P-body localization. We thus tested the effect of deleting it from the full-length sequence, but surprisingly, 4E-T-Δ694-716 . Further C-terminal truncations [4E-T-ΔA-(1-808) and 4E-T-ΔA-Δ(808-845)] did not localize to P-bodies in >80% of transfected cells, but also showed partial nuclear localization and were not used further.To test this, we examined the GFP-4E-T-ΔA protein truncated at a.a. 972 and 845. Both these mutants localized less efficiently to P-bodies than the parent GFP-4E-T-ΔA protein A. GFP-4ETo validate these results, we investigated 4E-T-GFP proteins lacking N-terminal regions. Truncated mutants whose ORFs started at a.a. 181 and 354 retained their ability to localize to P-bodies, similarly to full-length 4E-T, confirming that C-terminal sequences were required for localization while eIF4E-binding was not A. 4E-T-A, it wasThese results show that the sequences necessary for 4E-T residency in P-bodies are located in its C-terminus, downstream of a.a. 694. Moreover, two types of localization defective mutants were obtained—permissive  and nonpermissive (4E-T 1-694) for endogenous P-bodies.Supplementary Figure S3).We then wished to test the effect of wild-type and mutant 4E-T proteins on endogenous eIF4E distribution. First, using the yeast two hybrid assay, we verified the interaction between human 4E-T and eIF4E proteins. Mutagenesis of the three key residues of the eIF4E-binding site YXXXXLL led to complete loss of binding in yeast A. SimilaRenilla luciferase as the test mRNA with five 3′ UTR BoxB hairpins and firefly luciferase as the co-expressed control mRNA, lacking 3′ UTR elements. 4E-T expression constructs, and control constructs, contained a combined λN-HA (NHA) element or an HA tag alone upstream and in frame with the main ORF . After 48 h, lysates were prepared and assayed in the Dual Luciferase assay system, and RNA was extracted for qPCR of reporter mRNA levels, relative to GAPDH. Results are usually reported as ratios of RLuc/FLuc activities and mRNA levels in the presence of HA-tagged or NHA-tagged proteins.Next, we used the tethered function assay to ask what effect recruiting wild-type and mutant 4E-T to the 3′ UTR of a reporter mRNA had on its expression and stability. We used the λN-BoxB system, with main ORF A. TypicaSupplementary Figure S4). qPCR of the reporter mRNA levels showed that NHA-4E-T largely reduces expression of tethered mRNA at the level of translation  in global protein synthesis in the presence of GFP-4E-T but neither by GFP alone nor the Y30A 4E-T mutant . We provisionally concluded that ectopic 4E-T has two effects on protein synthesis, a general one affecting all translation, presumably by sequestering eIF4E, and an additional one on bound mRNAs. To assess this more thoroughly, we transfected three concentrations of NHA-tagged protein plasmids, which resulted in a wide range of expression as shown by western blotting . As expected, NHA-GFP and NHA-Pat1b, irrespective of levels, have little effect on the control firefly luciferase activity, while NHA-4E-T reduces its expression in a dose-dependent manner, as does NHA-4E-T ΔC, which contains the eIF4E-binding site but lacks the P-body localization sequence . Moreover, both NHA-4E-T and NHA-Pat1b significantly reduce the expression of Renilla-BoxB mRNA, in contrast to NHA-GFP. The panel showing the luciferase activity ratios confirms the previously reached conclusion that 4E-T and Pat1b both reduce expression of bound mRNA . By reporting the separate luciferase data, we further verify that 4E-T, unlike Pat1b, has both a general effect on protein synthesis translation, as shown by others  activities were reduced in cells transfected with HA/NHA-4E-T, though not by HA/NHA-GFP or -Pat1b proteins, while bound mRNA (RLuc) was significantly more inhibited only by NHA-4E-T and NHA-Pat1b (data not shown). Moreover, in y others ,22, and 4Lϕ on translation, having previously verified loss of eIF4E binding using GFP-Trap immunoprecipitation . In a subsequent experiment in which comparable levels of 4E-T proteins were expressed, the ratio of Rluc/Fluc activities  indicates that the eIF4E-binding mutant of 4E-T represses bound mRNA at least as well as the wild-type protein, if not significantly more . NHA-4E-T-Y30A served as an eIF4E-binding mutant control in this experiment, which did not affect Fluc mRNA activity but repressed tethered mRNA , any possible independent effects of the subdomains should have been detected, despite their low levels. To explore the possible role of the CHD, we removed this ∼ 25-amino acid-long highly conserved region from 4E-T , and found that it repressed tethered mRNA as well as the full-length protein . As 4E-T 1-694 repressed bound mRNA equally efficiently  containing the conserved CHD, and the C-terminal (602–985) domain encompassing the P-body localization signals. Neither of these domains affected bound mRNA translation significantly, though unlike the N-terminal fragment, the Mid- and C-ter domains were poorly expressed (data not shown). We note, however, that since tethered 4E-T repression is relatively protein concentration independent  complex followed by decapping and 5′ exonuclease action ,50. To elization A and B. lization C and D. lization F. In sumlization  and 7.FRenilla luciferase mRNA . These results mirror those we obtained previously in Xenopus oocytes with microinjected in vitro transcribed mRNA. We found that Acapped reporter mRNAs bearing the CSFV IRES were as robustly translated as capped mRNAs, and showed that Acapped CSFV IRES Fluc MS2 hairpin mRNA was unaffected by tethered MS2-4E-T, unlike the capped Fluc MS2 hairpin mRNA, which was repressed .To examine to what extent endogenous 4E-T regulates protein synthesis, we compared the rate of synthesis with  by ∼55% A, in linRenilla luciferase reporters lacking or containing two let-7 microRNA binding sites in their 3′ UTR . The result with LSm1, a P-body component required for P-body assembly , while other metazoa are restricted to a single 4E-T gene. Drosophila 4E-T is moderately expressed in various larval and adult tissues, including the central nervous system, with the highest levels seen in the ovary, while Drosophila Cup is highly expressed in both the ovary and testis (Flybase), indicating that the two paralogues may coexist, possibly to carry out distinct functions. The nematode 4E-T homologue Spn-2 gains its name from being spindle orientation defective. A mutant allele defective in normal spindle orientation in early embryos introduces a stop codon in place of Q at amino acid residue 540 , the truncation we defined as dominant negative for P-bodies.To identify the region that targets 4E-T to P-bodies, we reasoned that this would likely be common to previously characterized 4E-T homologues with similar roles in eIF4E binding, translational control of specific mRNAs and enrichment in mRNP. However we found very limited sequence homology between vertebrate 4E-T and Cup, and refocused our attention on Xenopus and mouse oocytes . We reasoned that this outcome was due to the sequestration of eIF4E by wild-type 4E-T, preventing translation initiation. Interestingly, RNAi knock-down of eIF4E does not affect global protein synthesis significantly, but rather, in a homeostatic mechanism, leads to the degradation of hypophosphorylated 4E-BP1 allowing any residual cap-binding protein to perform its function (4Lϕ)-independent manner. To our knowledge, this is the first report of a protein that can affect expression of both control and bound mRNAs, via distinct mechanisms.We observed, in reporter assays and in function . In cont4Lϕ-independent repression. However, we have recently shown that while human 4E-T indeed binds eIF4E2, as well as eIF4E1, mutagenesis of the critical tyrosine at position 30 to alanine, either alone or in combination with similar changes to the two leucine residues, fully prevented both interactions /eIF4B, eIF1 or eIF1A. 4E-T had no significant effects on the stability or polyadenylation of bound mRNA. However, when binding to eIF4E was prevented, modest effects on total levels were observed, involving enhanced binding to the CNOT deadenylase, followed, very likely, by enhanced decapping resulting from reduced protection of the cap structure by mutant tethered 4E-T.Drosophila Cup. Drosophila Cup possesses two binding sites for eIF4E, one being a canonical sequence, and the downstream one being ELEGRLR, but unlike the case with 4E-T, mutagenesis of the canonical site does not fully prevent interaction with eIF4E, while mutagenesis of the second site only partially prevents binding  site represses translation, in the absence of mRNA decay, but promotes deadenylation, as does wild-type Cup. On the other hand, mutagenesis of the second site resulted in the large-scale decay of bound mRNA, due to decapping in addition to deadenylation , mRNAs undergo partial poly(A) shortening rather than elongation  represses translation when bound to mRNA normally, presumably via 3′ UTR-binding proteins (rather than a tethering protein) remains to be investigated. Our results indicate that these putative RNA-binding proteins do not require the involvement of P-bodies, as repression by the tethered protein occurs in their absence, though we cannot exclude the participation of individual factors dispersed in the cytoplasm. In agreement, Cup lacks P-body localization sequences, and depletion of several P-body components from S2 cells did not relieve bound mRNA repression . Moreovether Cup  or 4E-T Drosophila CG32016/4E-T results in modest relief of a Vha68–1 reporter mRNA with two mir-9b binding sites in S2 cells  3′ UTR to repress its translation but this does not require P-granules as it is still observed in pgl-1 mutants , presumably RNA-binding proteins that naturally tether 4E-T to mRNA, and how they occlude ribosome binding. 4E-T and the related Cup proteins are particularly highly expressed in oocytes and ovaries in flies, worms and i et al.  recentlySupplementary Data are available at NAR Online, including [44].Wellcome Trust [084885/Z/08/Z] and BBSRC [BB/J00779X/1 to N.S.]; MRC [G0902052 to M.B.]; Gates Cambridge Scholarship postgraduate funding (to A.K.). Funding for open access charge: Wellcome Trust and BBSRC.Conflict of interest statement. None declared.


Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen  would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle. Antibodies play a vital role in preventing viral infection and offer protection against subsequent re-challenge, providing protective antibody titres are maintained. The maiAntigens that induce T-cell help to orchestrate a high affinity class-switched serological response are termed T-D antigens. During a T-D antigenic challenge, a small proportion of activated B cells differentiate into short-lived plasma cells within the T-cell regions of the secondary lymphoid organs and secrete low affinity antibodies for a short period of time. The rem+ T cell help, by cross linking multiple B cell receptors (BCRs) on the naïve B cell surface[Conversely, T-I antigens are able to initiate a serological response in the absence of T-cell help. There are two types of T-I antigens, type 1 are polyclonal B cell stimulants and type 2 are non-polyclonal stimulants. Type 2 T-I antigens possess highly organised, repeating structures that are able to activate naïve B cells in the absence of CD4 surface,12. A se surface,13 or co surface and in n surface,16.Many pathogens contain both T-I and T-D antigens, virus capsids that have a repetitive/non-random structure, such as foot-and-mouth disease virus, FMDV, with antigenic epitopes spaced 5–10 nm apart) tend to preferentially generate a T-I immunological response. Indeed,+ T-cells via cytokines[Upon re-exposure to a specific antigen or upon ytokines, from miytokines, or fromytokines. This meytokines, has nevIn the present study, we assessed and compared the kinetics of plasma and memory B cells generated in cattle immunised with either a T-D antigen  or type 2 T-I antigen . We also investigated whether a subsequent immunisation with a third party antigen exerted an effect upon the B cell response previously established to either the T-D or type 2 T-I antigen.Bos Taurus, Institute for Animal Health, Compton, UK) were split into two randomly selected groups of five. In the first group, each calf was subcutaneously immunised with 3 mg of the hapten 2,4,6-trinitrophenyl (TNP) conjugated to the T-D antigen chicken gamma globulin  in QuilA adjuvant . In the second group, each calf was subcutaneously immunised with 3 mg of TNP conjugated to the T-I antigen aminoethylcarboxymethyl-FICOLL  in QuilA adjuvant. All calves were boost-immunised 29-days later with the same immunising antigens, as described above (TNP-CGG in the T-D group and TNP-AECM-FICOLL in the T-I group). At day 134 post-primary immunisation (pi) all calves, from both groups, were subcutaneously immunised with 3 mg of tetanus toxin fragment C  in incomplete Freund’s adjuvant . Subsequently, all animals were boosted with TTC and IFA at day 197 pi. A group of control animals were given a primary and booster immunisation of adjuvant alone (including IFA). These animals showed no detectable antigen-specific plasma, memory B cells or IgG titres throughout the study. Heparinised peripheral blood and serum samples were taken from all animals at various time points following the immunisations. All experiments were approved by the Institute’s ethical review process and were in accordance with national guidelines on animal use.Ten male 8-month-old conventionally reared Holstein-Friesian calves . After two further washing steps using dPBS, the PBMCs were enumerated using a Neubauer haemocytometer . A fraction of the enumerated fresh PBMCs were diluted to the required concentration and stored at 4°C until required for the detection of plasma cells.Bovine PBMCs were isolated from the heparinised peripheral blood samples using density gradient centrifugation. The blood samples were diluted 1:2 using Dulbecco’s phosphate buffered saline , layered over Histopaque® 1.083 g/mL (Sigma-Aldrich) and centrifuged for 30 min at 1200 × 7 bovine PBMCs were cultured for 6 days in 20 mL of ELISPOT media  supplemented with 5 μg/mL pokeweed mitogen , 10 μg/mL anti-bovine CD40 mAb , 20 ng/mL recombinant human IL-2  and 7.3 units/mL bovine IL-10 . At the end of the culture period, the 6 day-cultured PBMC were recovered, washed twice in ELISPOT media, enumerated and stored on ice until being used for the detection of induced ASC by ELISPOT assay.An “ELISPOT medium” was used in all experiments aimed at quantifying plasma and memory B cells and consisted of RPMI 1640 with L-Glutamine and 25 mM Hepes  supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), 1× non-essential amino acids , 1 mM sodium pyruvate and 15% heat-inactivated horse serum (Sigma-Aldrich).At the indicated time-points, some PBMCs were cultured as described below to induce the terminal differentiation of memory B cells into antibody secreting cells (ASC) prior to their detection by ELISPOT assay. Briefly, 2 × 101/1000 Chicken serum  or 10 μg/mL TTC (PX’Therapeutics) in 0.1 M carbonate buffer (pH 9.6) for 2 h at 37°C. Plates were then washed 5 times in PBS and then blocked using 4% dried milk  in PBS. Following blocking, the plates were washed 5 times in PBS and stored at 4°C until required.MultiScreen™–HA plates  were coated with either 0.25 μg/mL TNP-Ovalbumin (TNP-Ova), 6 cells/mL and 1:2 serial dilutions were performed in ELISPOT medium down to 1.5 × 105 cells/mL. 100 μL/well of each suspension (in duplicate) was then incubated on the coated plates overnight at 37°C in a 5% CO2 incubator. Subsequently, the PBMCs were washed from the plates 5 times using PBS containing 0.05% Tween20 (Sigma-Aldrich). 100 μL per well of a 1/1000 dilution of sheep anti-bovine IgG conjugated to horseradish peroxidase  was then added to the plates for 3 h at room temperature. Following the incubation step, the plates were again washed 5 times using PBS containing 0.05% Tween20. Detection was performed by the addition of 100 μL/well of 3-amino-9-ethylcarbonate substrate  and subsequent incubation at room temperature for 1 h. The reaction was stopped by washing the plates with tap water and allowing them to dry overnight at room temperature.Fresh or 6 day-cultured PBMCs were suspended at 5 × 106 PBMCs for plasma cells and induced-ASC number per 106 cultured PBMCs for memory B cells (mean of duplicates). During the ELIspot data analysis any well with less than 2 spots was considered to be negative and were treated as having zero ASCs detected (minimum sensitivity: 4 ASCs per 1 × 106 PBMCs). There was very little variance (< 17%) shown between the duplicate observations within each animal.Enumeration of the red coloured spots on the dried ELISPOT plates was performed using the automated AID ELIspot reader . Each individual “spot” was used as an indicator for an ASC. The ELISPOT results were manually validated for false positives and expressed as ASC number per 101/50 dilution, and 100 μL per well of each diluted serum was added before incubating the plate for 1 h at room temperature. For determination of the plate background optical density (OD) values, some wells were incubated with blocking buffer alone. Following the incubation, the plates were washed and incubated for 1 h at room temperature after addition of 100 μL/well of 1/100,000 dilution of sheep anti-bovine IgG conjugated to horseradish peroxidase (AbD Serotec). Following washing, the plates were incubated for 10 min with 3,3’,5,5’-tetra-methyl benzidine  substrate at room temperature. The reaction was stopped by the addition of 50 μL of 1.2 M H2SO4. The OD values at 450 nm and 690 nm were determined, for each well using a Biotek ELx808 absorbance plate reader . The antigen-specific antibody titres were calculated as follows: the log10 values of sample dilutions were plotted against the log10 values of sample OD and a regression analysis was performed on the linear part of the curve allowing the endpoint titre to be calculated using an OD of twice the background. Results were expressed as the mean of duplicate determinations.96-well plastic plates  were coated for 1 h at room temperature with 100 μL/well of either 20 μg/mL TNP-OVA (Biosearch Technologies) or 10 μg/mL TTC (PX’Therapeutics) in carbonate buffer. Following incubation the plates were washed 5 times in PBS containing 1% Tween20 (Sigma-Aldrich). The plates were then blocked for 1 h using 100 μL/well of PBS containing 1% Tween20 and 4% dried milk (blocking buffer), followed by a washing step, as described above. Serum samples (in duplicate) were serially diluted 1:3 in blocking buffer starting at P > 0.05) terms as judged by likelihood ratio tests. Data were considered significant if the calculated P values were < 0.05.Data for the number of antigen-specific IgG ASCs (for plasma and memory B cells) were log + 1 transformed, then analysed using mixed linear models including time (as a factor) and vaccine (TNP-CGG or TNP-AECM-Ficoll), and an interaction between them as fixed effects and between animals as a random effect. Model selection proceeded by stepwise deletion of non-significant (n = 5) and approximately day 22 pi for the T-I group    and remained elevated until day 16 pi   before declining gradually  and boost  TNP immunisations  and the T-I (TNP-AECM-FICOLL) groups began to increase from day 6–8 post-primary TNP immunisation (pi) and peaked at approximately day 10 pi for the T-D group   . At day 34 post-TNP boost, we also assessed the B cell memory response to CGG using chicken serum as a positive control within the assay. CGG-specific memory B cells were only detected in the T-D group and were approximately 5–6 fold more numerous than TNP-specific memory B cells . In the T-I group, TNP- or CGG-specific plasma or memory B cells were not detected at any time-point following either the primary or booster TNP immunisation.In the T-D group, there were no detectable TNP-specific plasma cells post primary immunisation. However, a burst of TNP-specific plasma cells was observed at days 3–5 post-TNP boost  at day 134 pi and were re-immunised with TTC at day 197 pi.6 PBMCs, day 148 pi, n = 10)  . A small burst of IgG TNP-  and CGG- specific plasma cells was also observed in the T-D group at days 3 to 14 post-TTC primary immunisation . Another small burst of CGG-specific plasma cells was observed in 3 out of 5 calves at days 3 to 5 post-TTC boost  (p < 0.05) in the animals that had previously been immunised with the T-D antigen, TNP-CGG   and then gradually declining . There was also an increase in the number of TNP- and CGG-specific memory B cells in calves previously immunised with the T-D antigen at day 6–8 post-TTC boost   FigureA. Follow) FigureA (p < 0.n = 10) and day 7 post-TTC boost   as compared to day 0 post-TTC primary immunisation   as compared to day 0 post-TTC boost . In the T-I group; TNP-specific IgG titres were not increased following either the TTC primary or boost immunisation  following the immunisation of humans with an unrelated T-D antigen (tetanus toxoid)[Immunisation with the third party antigen (TTC) in this study resulted in similar TTC-specific plasma and memory B cell kinetics that have been previously described for other TD antigens,15,20,21 toxoid).There was a steady increase in the number of TTC-specific memory B cells post-TTC primary immunisation followed by at least a 14 fold increase in the number of these cells post-TTC boost. The T-D group also showed an increase in the number of TNP- and CGG-specific memory B cells only post-TTC boost (at day 6–8 post-TTC boost). Based on the timing of emergence of the TNP- and CGG memory B cells, it is unlikely that these memory B cells were generated from newly activated naïve B cells, but instead suggests that pre-existing TNP- and CGG-specific memory B cells were subjected to one (or more) round(s) of antigen-independent (bystander) proliferation within the T-cell regions of the draining lymph node. This bystander stimulation of T-D antigen-specific plasma and memory B cells may provide a mechanism for maintaining long term T-D antigen-specific antibody titres.In conclusion, the present study showed that bystander stimulation of an established T-D B cell memory response may occur in cattle, providing a possible mechanism of maintaining protective antibody titres for long periods of time. Our findings also provided a better characterisation of the key differences between T-D and T-I immune responses in cattle. However, as highlighted by Fink et al., more studies are required to further elucidate the mechanisms of the T-D and T-I2 activation of antigen-specific plasma cells. As demoThis knowledge will be particularly useful in elucidating the B cell response to the largely T-I antigen, FMDV. Indeed ASC: Antibody secreting cell; T-I: T-independent; T-D: T-dependent.The authors declare that they have no competing interests.CFJG carried out the majority of the immunoassays performed in this study, the data interpretation and drafted the manuscript. EAL participated in the design of the study, carried out some of the immunoassays and revised the manuscript. BVC performed some of the immunoassays. HP participated in the design of the study and study coordination. SG performed the statistical analysis. AJP participated in the design of the study, data interpretation and manuscript revision. CC participated in the study design and coordination. BC conceived the study and revised the manuscript. All authors have read and approved the final manuscript.


For the diagnosis of seasonal influenza, clinicians rely on point-of-care testing (POCT) using commercially available kits developed against seasonal influenza viruses. However, POCT has not yet been established for the diagnosis of pandemic influenza A virus (H1N1pdm) infection due to the low sensitivity of the existing kits for H1N1pdm.An immunochromatography (IC) test kit was developed based on a monoclonal antibody against H1N1pdm, which does not cross-react with seasonal influenza A or B viruses. The efficacy of this kit (PDM-IC kit) for the diagnosis of H1N1pdm infection was compared with that of an existing kit for the detection of seasonal influenza viruses (SEA-IC kit). Nasal swabs (n = 542) were obtained from patients with flu-like syndrome at 13 clinics in Osaka, Japan during the winter of 2010/2011. Among the 542 samples, randomly selected 332 were further evaluated for viral presence by reverse transcriptase polymerase chain reaction (RT-PCR). The PDM-IC kit versus the SEA-IC kit showed higher sensitivity to and specificity for H1N1pdm, despite several inconsistencies between the two kits or between the kits and RT-PCR. Consequently, greater numbers of false-negative and false-positive cases were documented when the SEA-IC kit was employed. Significant correlation coefficients for sensitivity, specificity, and negative prediction values between the two kits were observed at individual clinics, indicating that the results could be affected by clinic-related techniques for sampling and kit handling. Importantly, many patients  were prescribed anti-influenza drugs that were incongruous with their condition, largely due to physician preference for patient responses to questionnaires and patient symptomology, as opposed to actual viral presence.Concomitant use of SEA-IC and PDM-IC kits increased the likelihood of correct influenza diagnosis. Increasing the credibility of POCT is anticipated to decrease the inappropriate dispensing of anti-influenza drugs, thereby minimizing the emergence of drug-resistant H1N1pdm strains. Swine-origin pandemic influenza A virus (subtype H1N1pdm) emerged in April, 2009 and rapidly spread across the globe, becoming one of the most common human influenza A viruses in the world The H1N1pdm virus contains a triple-reassortant genome that includes a combination of avian, human, and swine influenza virus gene segments. The H1N1pdm genome encodes polymerase basic protein 2 (PB2) and polymerase subunit A (PA), both derived from the North American avian lineage; polymerase subunit B1 (PB1), derived from human seasonal influenza A H3N2; neuraminidase protein (NA) and matrix (M) proteins, derived from the Eurasian swine lineage; and hemagglutinin (HA), nucleoprotein (NP), and nonstructural (NS) proteins, derived from the North American classical swine lineage Based on epidemiological data from Mexico, where the estimated case fatality ratio in 2009 was 1.2% overall and 5.5% among individuals over 60 years of age Several groups are at elevated risk for H1N1pdm infection, including pregnant women, individuals with diabetes, and the obese, elderly, and very young Rapid immunologic diagnosis of H1N1pdm was attempted soon after its emergence in 2009 by using immunochromatography (IC) test kits previously developed for the detection of seasonal influenza viruses (SEA-IC kits). Nonetheless, these SEA-IC kits could not reliably differentiate H1N1pdm from seasonal influenza A viruses H1N1 and H3N2. Indeed, subsequent analysis via reverse transcriptase polymerase chain reaction (RT-PCR) indicated that existing SEA-IC test kits showed significantly low sensitivity for H1N1pdm Our previous research with clinical samples confirmed the efficacy of the newly-developed H1N1pdm IC test kit (PDM-IC kit) In the current study, the PDM-IC test kit was evaluated for the rapid diagnosis of H1N1pdm infection using clinical samples obtained during the 2010/2011 winter influenza season in Osaka, Japan. A total of 542 clinical samples collected from patients at 13 clinics were involved in the evaluation. The PDM-IC kit was assessed relative to a previously-developed SEA-IC kit for its accuracy and limitations of sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs). Subsets of the samples were also subjected to RT-PCR and virus isolation (VI) analyses. The data were then compared with clinical data regarding symptoms and drug prescriptions so as to determine the general applicability and reliability of rapid diagnosis kits for point-of-care testing (POCT) at clinics.The Prime Check Flu (H1N1) 2009  for the rapid detection of the NP protein derived from H1N1pdm Samples of nasal or nasopharyngeal fluid were collected from patients with influenza-like symptoms (n = 542) at 13 clinics (termed “A” through “M” clinics) under the guidance of the Higashinari-ku Medical Association in Osaka, Japan during the winter of 2010–2011 . Two swabs were taken from each patient; one sample was tested using the PDM-IC and SEA-IC kits, and the other was tested using RT-PCR and/or VI.2/95% air) at 37°C.The following virus strains were used: A/Suita/01/09 (H1N1pdm virus); A/New Caledonia/20/99 ; A/Wyoming/2/03 ; and B/Malaysia/2506/04 . Madin-Darby canine kidney (MDCK) cells were used for propagation of the viruses. The MDCK cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator  between the two kits or between the kits and RT-PCR. The additional 184/263 samples were randomly selected from the original 332 RT-PCR samples. To isolate the influenza viruses from the clinical samples, MDCK cells were inoculated with clinical specimens and incubated at 37°C in Dulbecco’s Modified Eagle Medium (DMEM) containing Nutrient Mixture F-12  and 1% v/v Antibiotic-Antimycotic liquid (Invitrogen), 0.4% w/v bovine serum albumin, and 2 µg/mL Trypsin Acetylated from Bovine Pancreas, Type V-S . Cell were incubated for 3 weeks, or until cytopathic effects were observed.Isolated viruses were identified by PAP staining using two influenza virus A subtype-specific murine monoclonal antibodies, C179 (H1-specific) and F49 (H3-specific), and the influenza virus B HA-specific murine monoclonal antibody, 7B11 , as described previously This study used the results of RT-PCR as the “gold standard”. Based on RT-PCR, the results of the test kits were divided into four groups, i.e., “true-positive (TP)”, “true-negative (TN)”, “false-positive (FP)”, and “false-negative (FN)” cases. Results of the SEA-IC kit were divided into TP, TN, FP, and FN cases, as follows: TP ; TN (“A−B−” with “−” by RT-PCR); FP (“A+B−” with “−” by RT-PCR); and FN . Results of the PDM-IC kit were divided into TP, TN, FP, and FN cases, as follow: TP (“pdm+” with H1N1pdm by RT-PCR); TN ; FP ; and FN (“pdm−” with H1N1pdm by RT-PCR). Statistical analysis was performed for sensitivity values, specificity values, positive predictive values (PPV), and negative predictive values (NPV) which were calculated according to the following formulas: sensitivity = TP/(TP+FN); specificity = TN/(FP+TN); PPV = TP/(TP+FP); and NPV = TN/(FN+TN). All results were assessed by correlation analysis, Student’s t-test, or the chi-square test, performed using SPSS version 18 . The level of statistical significance was set at P<0.05.The research protocol for the collection of human samples was approved by the Ethics Committee of the Research Institute for Microbial Diseases, Osaka University, Japan. Informed consent was obtained from all patients in writing before enrollment in the study.Swab samples (n = 542 duplicate samples) were simultaneously collected from 542 patients with influenza-like symptoms presenting at 13 clinics (clinics “A” through “M”) within Higashinari-ku, Osaka, Japan from November, 2010 through April, 2011 . The stuOf the two swab samples collected from each patient, one sample was immediately tested using the PDM-IC kit and the SEA-IC kit, and the other was kept at −80°C and subsequently used for RT-PCR and VI analyses. The PDM-IC and SEA-IC kits were designed for the rapid detection of NP proteins derived from the H1N1pdm virus and the seasonal influenza A and B viruses, respectively.The results from the SEA-IC kit were as follows: 44.7% (242/542) of the cases tested positive for influenza A virus; 3.5% (19/542) of the cases tested positive for influenza B virus; and the remaining 51.8% (281/542) of the cases tested negative for either influenza A or B virus . The PDMThe results of the RT-PCR analysis were as follows: 48.5% (161/332) of the cases tested positive for H1N1pdm; 8.1% (27/332) of the cases tested positive for seasonal influenza A virus H3N2; 4.8% (16/332) of the cases tested positive for influenza B virus; and 38.6% (128/332) of the cases tested negative for any type of influenza virus . No seasThe SEA-IC kit showed 89.2% sensitivity, 80.5% specificity, 87.9% PPV, and 82.4% NPV based on the data for influenza virus-positive cases obtained by RT-PCR (332 cases) . By contFurther analysis regarding sampling time after the onset of clinical signs was next performed. The accuracy of the results obtained using the SEA-IC kit significantly decreased for samples collected on the third or more day after onset (4.2±2.2 days) compared with those collected on the previous days . This waAs summarized in Further, the 263 samples, including the above-described samples showing inconsistent results between two IC test kits (n = 12), between the SEA-IC kit and RT-PCR (n = 47), and between the PDM-IC kit and RT-PCR (n = 20), were analyzed by VI. As summarized in To understand the relationship between clinical symptoms and IC test kit results, we used the combined results from the two IC test kits. Based on the results of POCT, the samples obtained from the patients were classified into five groups: 1) “A+B−” by SEA-IC and “pdm−” by PDM-IC for seasonal influenza A virus infection; 2) “A−B+” by SEA-IC and “pdm−” by PDM-IC for seasonal influenza B virus infection; 3) “A+B−” by SEA-IC and “pdm+” by PDM-IC for H1N1pdm virus infection; 4) “A−B−” by SEA-IC and “pdm+” by PDM-IC for possible H1N1pdm virus infection; and 5) “A−B−” by SEA-IC and “pdm−” by PDM-IC for the absence of influenza virus infection. Analysis of the relationship between clinical symptoms and POCT results revealed that there was a significant difference between the symptoms in influenza-positive and influenza-negative patients . For exaNo significant differences were observed in terms of other symptoms, such as fever, lower respiratory tract inflammation, arthralgia, or diarrhea. In particular, most of the patients with influenza-like syndrome who were enrolled in this study presented with a high fever, and there was no significant difference (P = 0.463) between patients diagnosed as influenza-positive (98.9%) versus influenza-negative (98.1%). As shown in Of 179 cases confirmed as influenza virus-positive by RT-PCR except 11 cases of “A−B−, pdm+” by the IC test kit results, 95.5% (171/179) were appropriately prescribed anti-influenza drugs based on positive IC test results. However, 8 influenza virus-positive patients out of 179 did not receive prescriptions due to negative IC kit results. An additional 47 cases were prescribed drugs despite influenza virus-negative RT-PCR results due to 18 false-positive SEA-IC kit results, six false-positive results stemming from both kits, and 23 true negative results stemming from both kits. On the other hand, 27 patients at six clinics were prescribed anti-viral drugs even though they tested negative for influenza virus by POCT. Of these 27 patients, three cases of H1N1pdm infection and one case of H3N2 infection were subsequently detected by RT-PCR. Importantly, seven patients who did not receive drug prescriptions because of negative IC results later tested positive for H1N1pdm (six cases) or H3N2 (one case) by RT-PCR.The present study evaluated a newly-developed PDM-IC kit for its ability to expedite POCT for the detection of H1N1pdm. The study employed 542 duplicate swab samples collected during the 2010/2011 winter influenza season in one ward of Osaka city and two types of IC test kits, the previously-developed SEA-IC kit and the newly-developed PDM-IC kit. Comparison of the data with those obtained by RT-PCR analysis using the same samples revealed that the PDM-IC kit was significantly more sensitive and specific for H1N1pdm than the SEA-IC kit.The establishment of POCT as a widely accepted method for the diagnosis of influenza depends on physician access to commercially available rapid diagnostic test kits. POCT can easily be performed with such kits within minutes for the detection of seasonal influenza A and B viruses, and the obtained results generally show quite high sensitivity and specificity for these viruses. However, detection of H1N1pdm by test kits that have been developed for seasonal influenza A and B viruses is unreliable, with comparatively low sensitivity and specificity In this regard, Choi et al. The results presented herein, using 332 swab samples further analyzed by RT-PCR, showed 92.5% sensitivity and 95.3% specificity for the PDM-IC kit, relative to 89.2% sensitivity and 80.5% specificity for the SEA-IC kit. The high sensitivity and specificity of this and other newly-developed IC test kits for the rapid diagnosis of H1N1pdm can potentially be attributed to the characteristics of the monoclonal antibodies and the cut-off points chosen by the kit development company/personnel. In other words, the company that develops each kit can adjust the sensitivity and specificity of the kit according to its intended use. Users of these test kits should therefore be educated to select the type of kit most appropriate for their needs.An advantage of the PDM-IC test kit described in this study is that it could be employed with the same swab samples as the SEA-IC kit. Therefore, we were able to obtain results from both test kits using the same samples at the same time. Thus, the PDM-IC kit can be used as a POCT tool to supplement SEA-IC kit data in clinics. Comparison of the data obtained at 13 individual clinics showed significant correlations regarding sensitivity, specificity, and NPV between the SEA-IC kit and the PDM-IC kit. This indicated that the corresponding scores of the IC kits were most likely affected by the skills of the medical and co-medical staff at each clinic. Therefore, increased application of rapid diagnosis test kits for infectious diseases necessitates training in their proper use, and not just selection of the appropriate test kit.No apparent differences were observed between different age groups in terms of the results yielded by the PDM-IC kit, while some inter-age differences were detected with the SEA-IC kit. Of note, the accuracy of the results of the SEA-IC kit apparently deteriorated at ∼3 days after the onset of clinical symptoms. Similarly, a previous report showed that the sensitivity of another IC test kit for H1N1pdm abruptly declined when using the swab samples collected from patients on the third day after the onset of fever Several cases (12/542 = 2.2%) in this study gave inconsistent results when analyzed with the two different IC test kits. In addition, the IC kit results and RT-PCR results were inconsistent for 14.2% (47/332) of the cases. Although both kits yielded several false-negatives and false-positives, overall, the PDM-IC kit demonstrated better specificity for H1N1pdm than the SEA-IC kit. IC kit-derived false-negatives  might be explained by the lower sensitivity of the IC kit compared with RT-PCR. On the other hand, there were several IC kit-derived false-positives . One possible explanation for their occurrence might be the fact that viral antigens might persist even after virus particles had been removed from body. Alternatively, the monoclonal antibody employed by the kit might react with non-specific antigens derived from the host or from other pathogens. When the samples that gave inconsistent IC kit versus RT-PCR were examined by VI, several cases were observed that tested positive by VI, but negative by RT-PCR, especially for H1N1pdm. Furthermore, several additional cases tested negative by VI, but positive by RT-PCR, especially in H3N2. This indicates the possible difficulty of PCR amplification of target H1N1pdm DNA in the former case and of VI of H3N2 in the latter case. Regardless of the cause, further improvements in IC kits are required because false-negative and false-positive results lead to misdiagnosis upon POCT.A total of 305 of 542 patients were treated with anti-viral drugs at 13 clinics in the present study. When the RT-PCR results were examined for 229 of these 305 drug-subscribed patients, we found that 23 individuals were given drugs even though they tested negative for the influenza virus by both IC test kits and RT-PCR. The drugs were dispensed because of patient responses to questionnaires regarding typical clinical symptoms of influenza, influenza infection in close relationships, friends, and/or colleagues, or an affected individual’s desire to receive anti-viral drugs within the first day of fever occurrence, typically at a clinic that is open over the weekend. The latter case is thought by the conditions for influenza virus detection in such febrile patients might simply be too early, leading to false-negative results upon POCT.Soon after the emergence of H1N1pdm, the virus was thought to be sensitive to oseltamivir (Tamiflu); however, several recent papers report an increasing incidence of drug-resistant H1N1pdm strains The future global status of influenza viruses, including the evolution of H1N1pdm and/or the appearance of new pandemic strains, is largely unknown. Zhu et al. reported a new reassortant virus in swine in 2010 Table S1Duration of days after disease onset categorized by each age group.(DOCX)Click here for additional data file.Table S2Antiviral drug prescription information from 13 clinics for influenza virus-positive cases confirmed by RT-PCR.(DOCX)Click here for additional data file.


Pseudoalteromonas sp. SM495. As a thermolysin family metalloprotease, E495 was found to have multiple active forms in the culture of strain SM495. E495-M  and E495-M-C1  were two stable mature forms, and E495-M-C1-C2  might be an intermediate. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to oligopeptides, but higher affinity and catalytic efficiency to proteins. The PPC domains from E495 were expressed as GST-fused proteins. Both of the recombinant PPC domains were shown to have binding ability to proteins C-phycocyanin and casein, and domain PPC1 had higher affinity to C-phycocyanin than domain PPC2. These results indicated that the domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore, improving the catalytic efficiency. Site-directed mutagenesis on the PPC domains showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.E495 is the most abundant protease secreted by the Arctic sea-ice bacterium  Sea ices, an important part of polar oceans, critically influence the productivity of the polar oceans, global energy budgets, and atmosphere-ocean interactions in the Arctic and Antarctic zones Pseudoalteromonas sp. SM495 is a protease-secreting bacterium isolated from the Arctic sea ice. E495, the most abundant protease secreted by strain SM495, is a cold-adapted metalloprotease of the thermolysin family (M4) Bacillus thermoproteolyticus, has been studied in detail and extensively applied for the synthesis of the artificial sweetener aspartame Pseudoalteromonas sp. SM9913 was reported to have two PPC domains in its recombinant mature form E495 precursor comprises 730 amino acids. Besides the signal peptide and the propeptide which are cleaved off after maturation, E495 contains a catalytic domain and two C-terminal PPC domains in its precursor Ps. sp. SM9913 and pseudolysin from mesophilic Pseudomonas aeruginosa PAO1 Our previous study showed that the recombinant E495 containing only the catalytic domain has better cold-adaptation ability compared to vibriolysin MCP-02 from cold-adapted deep-sea bacterium Ps. sp. SM495 was isolated from the sea ice core sample at the 122–124 cm depth from ice surface Escherichia coli DH5α and E. coli BL21(DE3) was purchased from Novagen (USA) and grown at 37°C on LB medium supplemented with ampicillin for the selection of transformants. C-phycocyanin (CPC) and allophycocyanin (APC) were extracted and purified from Synechocystis sp. strain PCC6803 with the methods described by Nies et al. 2 (FAGFA), FA-Gly-Leu-NH2 (FAGLA) and FA-Gly-Val-NH2 (FAGVA) from Bachem A .The sea ice core sample was taken from Canadian Basin, Arctic Ocean  during the 2nd Chinese National Arctic Research Expedition, 2003 summer. The strain Ps. sp. SM495 was inoculated in the fermentation medium previously described Strain Procise 491 protein sequencer . The molecular weights (MW) of the proteases were determined using an Ultraflex MALDI-TOF/TOF mass spectrometer .The proteases were transferred into 12.5% gel using SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane  as described by Matsudaira SDS-PAGE was performed according to the method of Laemmli Ps. sp. SM495 in five kinds of media with different nitrogen sources was analyzed. Flasks were prepared with the fermentation medium previously described Ps. sp. SM495 was inoculated in the media and cultivated in a 50/500 ml flask at 15°C, 200 rpm for 90 h. After cultivation, the culture was centrifuged at 10,000× g, 4°C for 20 min. Fifteen microliters of each culture supernatant were subjected to zymogram to analyze the relative content of proteases in each culture.Protease production of To analyze the stability of E495 in the culture, the supernatant of the fermentation solution was incubated at 15°C for 15 min, 1 h, 3 h, 5 h, 10 h or 20 h. After incubation, the proteases in the solutions were analyzed with zymography. Zymography was performed as described above. To analyze the stability of the purified E495-M-C1 and E495-M, E495-M-C1 (0.05 mg/ml) and E495-M (0.04 mg/ml) were incubated at 4°C for 0, 5, 10, 20 d. After incubation, the samples were subjected to 12.5% SDS-PAGE to analyze the relative content of E495-M-C1 and E495-M in these samples.The protein concentration was determined using the Bradford method with bovine serum albumin  as a standard. The protease activity to casein was determined with the method described previously EcoRI/XhoI-linearized pGEX-4T-1 vectors  to construct plasmids  for expression of GST-fused PPC domains. All the expression plasmids were transformed into E. coli BL21(DE3) competent cells. Expression was induced with 0.2 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) at 15°C for 16 h. The GST-fused PPC domains  were purified by glutathione-agarose chromatography .The PPC domains  of E495 were amplified using PCR with previously constructed plasmid pET-22b-E495 as a template E. coli BL21(DE3). After confirmed by enzyme digestion and nucleotide sequencing, all mutations were expressed and purified with the same condition as GST-fused PPC domains.Site-directed mutagenesis was carried out by overlapping extension-PCR with the expression plasmids constructed above  as templates. Mutated sites  were introduced by the primers with single mutation . The mutSubstrate binding of GST-fused PPC domains was assayed using the “pull-down” affinity head interaction method described by Gary The interactions of PPC1, PPC2 and their corresponding mutations with CPC were analyzed at 25°C using a BIACORE 3000 instrument . Biacore CM5 research grade sensor chips, Tween 20, and Biacore amine coupling kit were purchased from GE Healthcare (USA). CPC was immobilized on the CM5 chip using standard amine-coupling method ka, kd and KD) from the raw data were calculated by non-linear regression or equilibrium binding analysis using the BIAevaluation software version 4.1  supplied with the instrument.To analyze the kinetic constants, various concentrations of the PPC domains  were passed over the chip surface at a flow rate of 30 µl/min. The surfaces were regenerated by injection of 20 mM NaOH at a flow rate of 30 µl/min prior to the next injection cycle. Kinetic constants (2 dmol−1) at the entire wavelength using the relation: [θ] λ = θλM0/10/c.CD spectra of the purified GST-fused PPC domains and their mutants in the same concentration (0.1 mg/ml) in 20 mM boric buffer (pH 8.0) were measured on a Jasco J810 spectropolarimeter (Japan) with a bandwidth of 2 nm, a response time of 1 s, and a scan speed of 200 nm/min. Each spectrum was an average of three scans monitored between 198 and 250 nm. The path length of the cuvette was 0.1 cm. The raw CD data were converted into mean residue ellipticity , a propeptide, a catalytic domain and two PPC domains . It can be concluded from their N-terminal sequences that the three forms of E495 all contain the catalytic domain with or without the PPC domains. Since the largest form has a MW of 55 kDa  towards FAGFA and the lowest towards FAGVA , indicating that PPC1 had higher affinity to CPC than PPC2. These results showed that the PPC domains in E495 have protein substrate-binding ability.In order to test whether the PPC domains of E495 has binding ability to protein substrate, the PPC domains of E495, PPC1, PPC2 and PPC12 (containing PPC1 and PPC2), were expressed as GST fusion proteins. The “pull-down” affinity head interaction assay was used to determine the binding ability of these fusion proteins to CPC and casein. As shown in The sequences of the PPC domains in some bacterial metalloproteases were aligned to analyze the conserve amino acid residues . Since tPs. sp. SM495 is a metalloprotease of the M4 family. E495 and some other proteases in this family, such as MCP-02 from Ps. sp. SM9913 and EmpI from Ps. sp. strain A28, have two C-terminal PPC domains in their precursors. Both MCP-02 and EmpI have one form of mature enzyme which contains only the catalytic domain The protease E495 secreted by the Arctic sea-ice bacterium Vibrio vulnificus metalloprotease was reported to be essential for efficient attachment to insoluble protein substrates and erythrocyte membranes Although PPC domains are found in the precursors of many metalloproteases and serine proteases, there are very few mature proteases containing PPC domains Many hydrolytic enzymes have a substrate binding domain (SBD), such as the cellulose-binding domain in cellulases Ps. sp. SM495 even in five different culture media, which may imply that E495 is the most abundant extracellular protease of Ps. sp. SM495 in the natural sea ice environment. The bacterial extracellular proteases in the Arctic sea ice ecosystem are mainly responsible for organic nitrogen degradation, whose mechanism is still unclear. Our results show that E495 has evolved high specific activity to many kinds of proteins, especially algal proteins, to be adapted to the sea ice environment. Moreover, E495 has more than one mature forms for protein degradation, which may be a strategy of strain SM495 to efficiently degrade the peptides and proteins in the sea ice. Therefore, as the most abundant extracellular protease of Ps. sp. SM495, E495 would play an important role in sea-ice organic nitrogen degradation and bacterial nutrition. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.Because sea ice environment is difficult to simulate under laboratory conditions, many researches about sea ice bacteria secreting enzymes are cultured using synthesis or fermentation media under laboratory conditions Figure S1Molecular weight of E495-M-C1 determined by mass spectroscopy. The molecular weight of E495-M-C1 was determined by using a Ultraflex MALDI-TOF/TOF mass spectrometer . The result showed that the molecular weight of E495-M-C1 is 43914 Da.(TIF)Click here for additional data file.Figure S2Comparison of the relative catalytic efficiency of E495, MCP-02 and pseudolysin to proteins CPC (A), APC (B) and gamma globulin (C). Each protease (0.3 µmol/L) was incubated at 37°C with CPC for 15 min, APC for 30 min, and gamma globulin for 12 h, respectively. Then, the hydrolysis products were analyzed on 12.5% SDS-PAGE gel. CPC and APC both contain two subunits α and β as shown in the figures.(TIF)Click here for additional data file.Figure S3Non-linear fit curves for α-casein hydrolysis by E495-M and E495-M-C1. The initial reaction rates were determined with 0–0.6 mM α-casein at 50°C.(TIF)Click here for additional data file.Figure S4Ps. sp. A28, the putative metalloproteinase from Ps. tunicata D2, and the metalloprotease II from Ps. piscicida. Identical residues are indicated by asterisks. Sequences were aligned using Clustal ×1.83.Alignment of the PPC domains of E495 with those of the metalloprotease from (TIF)Click here for additional data file.Figure S5CD spectra of GST-fused PPC domains and their mutants. The spectra were measured on a Jasco J-810 spectropolarimeter (Jasco Japan) with the method described in “Materials and Methods.”(TIF)Click here for additional data file.Table S1Oligonucleotide primers used to amplify PPC domains and their mutants.(DOC)Click here for additional data file.Table S2a.Quantitive analysis of the amount of CPC or casein bound by the PPC domains in the assay of the “pull-down” affinity head interaction(DOC)Click here for additional data file.


BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes,  BLM, WRN and other members of the RecQ helicase gene family play important roles in many aspects of DNA metabolism, in particular, during replication and repair of DNA double-strand breaks (DSBs) (s (DSBs) ,2. DSB rs (DSBs) .In vitro BLM and WRN are able to unwind structures that resemble HR intermediates (D-loops and Holliday junctions) and G-quadruplex structures at array ,10, whicat array . WRN is at array ,13. It iat array . In mousat array  and thisat array ,15. The at array , it bindat array  and it cat array ,19.A proportion of all tumours, in particular sarcomas, utilize a recombination-based mechanism known as alternative lengthening of telomeres (ALT) to maintain telomeres 20,. The eviSaccharomyces cerevisiae, as the yeast RecQ helicase (SGS1) is required for ALT-type survivors . W-V was derived from a normal fibroblast cell line from a 45-year-old male patient with Werner’s syndrome that was transformed by SV40 infection ,44. The 2O containing tRNA  so that on an average, each polymerase chain reaction (PCR) produced a single amplicon from one telomere molecule (50–80 pg/PCR). Each STELA comprised a 20 μl reaction containing 1× PCR buffer, 0.5 μM of the allele specific and teltail primer and 1 unit of a mixture of Taq (KapaBiosystems) and Pwo (Roche) polymerase (10:1). The amplification was performed as follows: 25 cycles at 96°C for 20 s, 67°C for 30 s and 68°C for 10 min. STELA products were size separated in a 0.8% agarose gel, transferred to a nylon membrane and hybridized to a 32P-labelled telomere probe. Labelled products were detected using a PhosphorImager  and analysed using the Image Quant v.2005 software.Allele-specific single molecule (sm)-STELA was used to amplify single telomeres at Xp/Yp or 12q using the XpYp-415C primer or the 12qSTELA primer (5′-CGAAGCAGCATTCTTCTCAG-3′) ,45,46. sThe detection of Xp/Yp or 12q telomere mutations was performed using sm-STELA products as templates for Telomere Variant Repeat mapping by PCR (TVR-PCR) to determine the distribution of TTAGGG and sequence-variant repeats ,45,47. TUCACAAGGAAUGAGAAAUAUUCAAGAGAUAUUUCUCAUUCCUUGUGAUU5′, and the shRNA sequence is shown in bold (target is in exon 17). The shBLM (3 μg) was linearized and transfected into (5 × 105) cells using electroporation . The media was supplemented with Geneticin  at a final concentration of 500 mg/ml 48 h after transfection. Selected colonies were grown for approximately 20 population doublings (PDs) before DNA and proteins were extracted for analysis. Control clones were generated by transfecting the cell lines with linearized pSuperior.neo.gfp vector (empty vector)  containing a DNA sequence for producing a short hairpin RNA (shRNA) against human BLM was constructed . The DNA oligo cloned into the plasmid was 3′ vector) .6) were lysed in sodium dodecyl sulphate (SDS)-lysis buffer  supplemented with 1× protease inhibitor cocktail (Sigma), 2 mM sodium orthovanadate and 2.5 mM sodium fluoride for 20 min at 4°C. Total protein extracts (50 mg) were resolved in 6% SDS-polyacrylamide gel electrophoresis for 1 h at 150 V and transferred to a nitrocellulose membrane. The membrane was incubated for at least 2 h with an antibody against BLM  or 1 h for the loading control β-Actin antibody . Horseradish peroxidise-conjugated secondary antibodies  were used to detect primary antibodies. Bound antibodies were detected using the enhanced chemiluminescence (ECL) plus western blotting detection system . The membrane was exposed to X-ray film and images analysed using the ImageJ software.Cells . The DNA used as template in the MVR-PCR was either a mutant or a progenitor molecule amplified previously by small-pool PCR.The interspersion of sequence variant repeats across the MS32 repeat array was determined by minisatellite variant repeat mapping by PCR (MVR-PCR), as described previously . In brieSmall-pool PCR was used to amplify MS1 and B6.7 minisatellite using 0.2 μM of primers MS1-420 with MS1+280 (50) and primers 67A with 67B , respectWRN at telomeres in ALT+ cells, we first determined the profile of telomere mutations in an SV40 transformed and immortalized ALT+ cell line derived from a donor with WS . Mutell line ,47,52. Tell line ) locatedell line ,54. The P = 0.000003, Fisher exact test 2-tail).Mutations at telomeres in ALT+ cell lines have been described previously  with an µ = 0 (0/450); Hilda Pickett and N.J.Royle., unpublished data). This mutation frequency is significantly different from that seen in the W-V cell line  but in line with other primary cells lines . Three telomeres  were analysed in each of 150 clonally derived DNA samples from the AG03141B+hTERT cell line, and no mutations were identified . Although this mutation frequency is higher than those measured in normal or telomerase+ cell lines  indicates that BLM is important for cell growth in ALT+ cells. This has been reported previously by Temine-Smaali et al. who showed that RNA interference-mediated depletion of BLM reduced the growth rate of U2OS (ALT+) cells but did not have a significant effect on HeLa   and U2OS WRN+; A. The null line) . Others ll line)  but to aNext, the shBLM plasmid or empty vector control was transfected into W-V cells and clones were isolated and expanded. After 20–21 PDs, the level of BLM expression was determined by western blot analysis. Most of the isolated clones showed very little or no BLM down-regulation. However, one clone (W-V-shBLM clone 6) showed a reduction of ∼60% B and twoAs stated earlier in the text, ALT+ cell lines and tumours are characterized by extraordinary instability at the MS32 minisatellite (D1S8) on chromosome 1 30,. This mi−3, Supplementary Figure S2) and in line with other telomerase-expressing immortal cell lines (30). Therefore, the absence of BLM alone does not result in high MS32 instability. We then determined the average mutation rate at MS32 in six empty vector control clones derived from the W-V cell line and compared it with the average mutation rate from three clones transfected with the shBLM plasmid, W-V-shBLM clones 6, 2 and 8, which showed varying levels of BLM depletion . Neither minisatellite showed instability. Therefore the data indicate that BLM specifically suppresses MS32 instability in ALT+ cells.We checked MS32 stability in the BLM-negative GM03403 lymphoblastoid cell line, the mutation frequency was low , and three fairly stable MS32 bands  were detected in the W-V cell line composed of 54, 106 and 126 repeats . MVR mapThe five mutant MS32 molecules analysed from the W-V-shBLM clone 6 contain expanded tracts of e-type repeats and mutants 2–5 are interrupted by a block of E-type repeats. It is difficult to relate the block-like structure of these mutants directly to the mapped common alleles in the W-V cell line, though allele 3 contains the longest homogenous array of e-type repeats. It seems that following depletion of BLM, the MS32 repeat array has undergone further homogenization and expansion  or perhaps break-induced replication (BIR) between sister chromatids ,62.−3 per PD, comparable with the telomere mutation rate obtained in WV-shBLM clone 8  than that seen in shBLM clones. The effect on telomere mutation rate in W-V-shBLM clone 8 was unexpected, as the clone showed only modest down-regulation (∼20%) when it was measured , though this was not seen in the W-V cell line or in the W-V-Control clone 4. We ruled out a problem of contamination since the single molecule STELA products used for TVR-PCR mapping were from different sized amplicons, showing they come from different telomere molecules. The repeated isolation of mutant molecules with the same interspersion pattern could arise from somatic mosaicism, from independent mutation events or a combination. For example, W-V-shBLM clone 6 mutant 1 was detected in 14 times . Thus BLM depletion in this WRN-ALT+ cell line (W-V) specifically increased the rate of mutation processes that remove variant repeats (as seen at MS32) and so tended to homogenize the repeat array towards pure (TTAGGG)n.In summary, the telomere mutation rates in W-V-shBLM clones 6 and 8 cannot be determined exactly, because of the unknown contribution of somatic mosaicism, but the data presented show the telomere mutation rate increased when BLM was down-regulated. In addition, mutations that resulted in homogenization of the telomere repeat array occurred much more often in the BLM-depleted clones  than in the control or W-V parental cell line  suggesting it is not contributing to the altered telomere mutation profile in the W-V cell line. Alternatively, a gene that regulates BLM activity may have altered expression, for example, HSP90 that inhibits BLM helicase activity in ALT+ cells .Supplementary Figure S5). In addition, Muntoni et al. showed that a related IIICF/c ALT+ cell line supports telomeric intra-allelic DNA copying . The mutant MS32 molecules in the BLM-depleted W-V clone  [G0500336 to N.J.R.]; Conacyt, Mexico (to A.H.-B.). Funding for open access charge: MRC.Conflict of interest statement. None declared.


Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans.A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus  A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses Agrobacterium tumefaciens Vir system is considered to be the paradigm of Type-IVA, which consists of 12 components, VirB1−VirB11 and VirD4 Legionella pneumophila and is composed of 25 genes on two separate regions. Region I contains seven genes , and Region II contain the other 18 genes  Coxiella burnetti Hin1056 of Haemophilus influenzaBased on a number of characteristics, including, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several subgroups: Type-IVA, Type-IVB systems and other T4SSs Streptococcus suis (S. suis), a GI-type T4SS-like system was identified in a new pathogenicity island (PAI) with a length of 89 kb S. suis, a Gram-positive species of Streptococcus found in pigs, has recently caused a rash of human infections in China and gained public attention S. suis isolates related to two recent large-scale outbreaks of human infection in China, we identified this new 89 kb PAI with GI-type T4SS-like system S. suis recipients through the 89K-encoded GI-type T4SS Streptococcus. This system is movable with the help of transposon factors (such as Tn916), which could also mediate the conjugative transfer of DNAs and enhance bacterial pathogenicity.T4SS was once believed to be present only in Gram-negative bacteria but has been found in Gram-positive organisms as well. Our previous work support that in Gram-positive species of Streptococcus strains used in the current study are listed in Supplemental Tables. Whole genome sequences of 50 strains of Streptococcus (ftp://ftp.ncbi.nih.gov/genomes). Draft genome sequences of 67 Streptococcus strains were also obtained from the NCBI database (The tococcus  were dowdatabase .virB1−virB11 and virD4) from the NCBI and UniprotKB database We manually collected 839 gene sequences of the 12 components of T4SS (http://t4ss.bioinfo-icdc.org/). This program combines the alignment algorithm approaches, prediction of protein functions, and domain evaluation to detect candidate T4SS genes (virB1−11 and virD4) in genomes of bacteria (the detailed method is shown in the Protocol S1) with good precision.To search for genes in the T4SS, a new program (TFSP) was used (virB/D genes), we found virB/D clusters in genomes of Streptococcus. In this study, determination of virB/D clusters conformed to the following criteria: (1) the distance between two nearby virB/D genes is less than 5 kb, (2) the total length of the virB/D cluster is less than 50 kb, and (3) the number of virB/D genes in a virB/D cluster is ≥3.Based on the locations of identified T4SS genes  was identified in Gram-positive strain is study . Differeis study .Streptococcus, we used bioinformatic methods to predict T4SS genes (Streptococcus strains with published genome sequences (Streptococcus strains (S. suis BM407 (NC_012926) has two copies of T4SS, while the other 13 genomes have only one. All 15 Type-IVC secretion systems own clustered virB1-like/virB4/virB6/virD4 or virB4/virB6/virD4 genes in the same direction and order  with Type-IVC secretion system by co-lineage comparisons genomic comparison. Compared with the 89 kb PAI of S. suis, the structures of these GIs are significantly variable , they still work in same three fields  ΔvirB4-89K, ΔvirB6-89K and ΔvirD4-89K were used as donors. And the transfer frequency of ΔvirB1-89K (9.2×10−7) was six-fold less than that of wild type strains The function of these four proteins  for the secretion of DNA has been proven by making individual knockouts of T4SS gene homologues rpoB clearly showed that Type-IVC secretion system is movable, for strains with and without Type-IVC secretion system co-existing in several branches  were identified with Tn916 transposons (S. pneumoniae CGSP14 and S. pyogenes MGAS2096) harbored Tn916 residues, whereas the other two strains  had Tn916 at another position on the chromosome . Further, it is movable with the help of transposon factors, which could mediate the conjugative transfer of plasmid DNA/transposons and enhance bacterial pathogenicity.In this paper, we present evidence that the GI-type T4SS-like system we experimentally defined earlier in Figure S1virB1–virB11 and virD4 genes were identified. The blue columns are the number of Streptococcus strains with virB/D genes, whereas the red columns are the number of Streptococcus strains with virB/D clusters.Strain numbers in which (DOC)Click here for additional data file.Figure S2Streptococcus compared to the 89-kb GI in S. suis 05ZYH33. GC%, locations of the virB/D genes, Tn916, and other important genes in GI are shown. A) S. suis SC84; B) S. suis BM407 (1000794∼1091159); C) S. suis BM407 (499472∼585444); D) S. pneumoniae P1031; E) S. pneumoniae G54; F) S. pneumoniae ATCC 700669; G) S. pneumoniae CGSP14; H) S. agalactiae 2603V R; I) S. agalactiae NEM316; J) S. pyogenes MGAS2096. Genes with varying functions are presented in different colors.Genome islands with T4SS in 10 strains of (DOC)Click here for additional data file.Table S1virB/D clusters identified in 14 Streptococcus strains. ID: virB/D cluster ID in a genome; S: start site of virB/D gene in genome; E: end site of virB/D gene in genome; D: direction of virB/D gene.List of (DOC)Click here for additional data file.Table S2Streptococcus strains with draft genomes used in this study. “+” indicates that there is an identified virB/D gene cluster in this strain.List of (DOC)Click here for additional data file.Protocol S1Detailed (DOC)Click here for additional data file.


PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes  The Hedgehog (HH) signaling pathway is fundamental during embryonic development, and is also implicated in the growth of a variety of tumors SUFU have been found in medulloblastoma SUFU loss of heterozygosity was observed in rhabdomyosarcoma (RMS) SUFU is a tumor suppressor gene.Loss of function of mammalian SUFU leads to ligand-independent activation of the HH pathway SUFU is localized on chromosome 10q24–25, and contains 12 exons SUFU , together with a GLI reporter construct. SUFU-FL was found to have a stronger effect than SUFU-ΔC in reducing GLI activity , and therefore characterized by a constitutively active HH signaling pathway, were used. The results indicated that the maximal repression was observed with SUFU-FL acting on GLI1FL, as seen with NIH3T3 cells. SUFU-ΔC, on the other hand, was found to be more effective than SUFU-FL in repressing the GLI1ΔN variant  were individually introduced into the RMS13 cell line. Transfection of the shRNAs did not alter the SUFU-FL expression levels, as determined by real-time RT-PCR analysis, highlighting their specificity for SUFU-ΔC . AdditioHuman SUFU plays an essential role during both normal development and cancerous transformation. SUFU is known to functionally regulate the GLI proteins, the ultimate effectors of the HH pathway Although both SUFU isoforms could be detected at the mRNA level, only low levels of the endogenous SUFU-ΔC protein were observed in the Western blot analysis of the RMS cell lines. This indicates that the SUFU-ΔC is less stable than SUFU-FL. The analysis of the heterologous expression of the SUFU variants in the Hek293 cell line is in agreement with a reduced stability of SUFU-ΔC relative to SUFU-FL in human RMS cells. Moreover, treatment with the proteasome inhibitor MG-132 confers a selective increase of both the endogenous and exogenous SUFU-ΔC protein, however, the levels of SUFU-FL are not reached. Consequently, one has to hypothesize that other mechanisms may also have a role in the reduced steady state levels of SUFU-ΔC, possibly lysosomal autophagy as recently implicated in Wnt signaling components Sufu−/− MEFs, SUFU-FL elicited a stronger inhibition than SUFU-ΔC on GLI activity , and Sufu−/− mouse embryonic fibroblasts (MEFs) were established by S. Teglund in our laboratory The NIH3T3 cell line, the Hek293 cell line, the alveolar rhabdomyosarcoma (A-RMS) cell line RMS13 and the embryonal rhabdomyosarcoma (E-RMS) cell line RD were purchased from ATCC , the E-RMS cell lines JR-1 and Rh36 were kind gifts from P. Houghton , CCA was a kind gift from P-L Lollini , and CT-TC a kind gift from H. Hosoi  followed by cDNA synthesis with random (N6) primers  and Superscript II . Real-time RT-PCR was performed with Power SYBR Green  on a 7500 Fast real-time PCR system (Applied Biosystems) with the following primers used; SUFU–Forward: Proteins were extracted and separated on 15% or 7% SDS-PAGE essentially as described 5′-GCTTCTAGAATAGGTTCAGAGTTGT and 5′-ACCAATCAACCCTCAGCGGCAGAAT. The SUFU-FL-FLAG and SUFU-ΔC-FLAG constructs were generated from the SUFU-FL-EYFP plasmid. Following BspE1/Xba1 double digestion, ligation of PCR products generated by the primer sets SUFU-Forward 5′-ACCAATCAACCCTCAGCGGCAGAAT and either FLAG-SUFU-FL-Reverse 5′-GCTTCTAGACTACTTGTCATCGTCGTCCTTGTAATCGTGTAGCGGACTGTCG or FLAG-SUFU-ΔC-Reverse 5′- GCTTCTAGACTACTTGTCATCGTCGTCCTTGTAATCGAGTTGTAACCAGGGT were performed. For all expression constructs the pCMV vector was used. The expression constructs and the appropriate reporter construct were transfected into cultured cells using FuGENE6  or Lipofectamine 2000 (Invitrogen). Expression of the SUFU variants in E.coli is described in Expression and reporter constructs have been described elsewhere Luciferase reporter assays were performed essentially as described previously Hek293 cells were plated in 4-well poly-d-lysine-coated dishes, and transfected 24 hours later with Myc-tagged SUFU constructs and GLI1FL-FLAG. 48 h after transfection, cells were fixed, permeabilized, incubated with anti-FLAG (Sigma-Aldrich) and/or anti-Myc  overnight and fluorescently labeled with Alexa Fluor dye-conjugated secondary antibodies . Imaging of the immunostained cells was performed using a Zeiss LSM510 confocal microscope. Individual experiments were performed at least in triplicate and the data from a representative experiment are shown.Two shRNA constructs (sh4 and sh5) targeting SUFU-ΔC were designed using sequences from SUFU exon 10a and the Insert Design Tool for the pSilencer Vectors . As a negative control, a shRNA targeting the Enhanced Green Fluorescent Protein (EGFP), shEGFP, was also designed. Complementary oligonucleotides with BamHI and HindIII sites at the ends were ordered from Sigma-Aldrich with the following sequences; sh4: 5′-CCTATCCTCGGAGCTCTGCTTCAAGAGAGCAGAGCTCCGAGGATAGGTT, sh5: 5′-TTTTCAGCAGCTCAAGAACTTCAAGAGAGTTCTTGAGCTGCTGAAAATT, shEGFP: 5′-GAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTCAT and cloned into the pSilencer 4.1-CMVpuro vector. All constructs were verified by sequencing. RMS13 cells were transfected with the shRNA constructs and subjected to puromycin (1 µg/mL) selection for 4 days, followed by RNA isolation and real-time RT-PCR.Figure S1Heterologous expression of SUFU variants in E. coli. SUFU-FL (upper panel) and SUFU-ΔC (lower panel) constructs were purified from E. coli as described above. Chromatograms of the last purification step, size-exclusion chromatography, are shown. Open arrows indicate peaks corresponding to protein eluted with the void volume (aggregated form), while filled arrows indicate peaks corresponding to monomeric (soluble form) protein fractions. Note the decreased amount of SUFU-ΔC relative to SUFU-FL in the soluble fraction.(PDF)Click here for additional data file.Figure S2The Western blot of  was stripped and incubated with a GLI2 antibody. Note that the levels of endogenous GLI2 remain unchanged irrespective of the introduction of SUFU-ΔC or SUFU-FL.(PDF)Click here for additional data file.Figure S3Western blot analysis of SUFU protein levels after shRNA down-regulation. shEGFP, sh5 or sh4 constructs were transfected into RMS13 cells, followed by SDS-PAGE gel electrophoresis and Western blot analysis by a SUFU antibody. Both a short and a long exposure are shown. SUFU-ΔC is indicated by an arrow. Vinculin was used as a loading control. A quantification of the SUFU-ΔC protein levels relative to SUFU-FL is shown below the blot, revealing a 33% reduction of SUFU-ΔC by sh5 RNA treatment.(PDF)Click here for additional data file.Methods S1Heterologous protein expression in E.coli and purification.(PDF)Click here for additional data file.Table S1Sequence of PCR primers.(PDF)Click here for additional data file.