Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide Ssuis . The objective of this study was to characterize the function of Ide Ssuis in the host-pathogen interaction. Edman-sequencing revealed that Ide Ssuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide Ssuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide Ssuis . Furthermore, expression of Ide Ssuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide Ssuis . Bactericidal assays confirmed a positive effect of Ide Ssuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide Ssuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
Streptococcus (S.) suis colonizes different mucosa of pigs, its main host. Virulent strains might, however, cross the mucosal barrier, cause bacteremia and infect various tissues leading to severe pathologies, such as meningitis, arthritis, endocarditis and serositis. Suppurative meningitis caused by S. suis is one of the most important diseases in modern swine production as it is associated with severe economic losses. S. suis exhibits a high degree of diversity among and within different serotypes. Serotype 2 is worldwide the most important serotype isolated from affected tissues in piglets and also an important zoonotic agent [1-3].
Numerous proteins involved in interaction with the host have been functionally characterized [4,5]. Recently, we identified a 124 kDa large Immunoglobulin M-degrading enzyme of S. suis, designated Ide Ssuis . The N-terminal region of Ide Ssuis is homologous to the 38 kDa IgG specific endoprotease IdeS (also known as Mac-1) expressed by S. pyogenes and sufficient for IgM cleavage. Ide Ssuis is a highly specific IgM protease expressed by all investigated S. suis strains, which included strains from four different serotypes and clonal complexes. Importantly, it is so far the only known protease cleaving specifically the intact IgM multimer. The specificity of this protease is underscored by several findings: (i) Ide Ssuis does not degrade porcine or human IgG or IgA, (ii) IgM of pigs but not IgM of any other investigated species is cleaved and (iii) incubation of different body liquids with Ide Ssuis , including cerebrospinal and joint fluids from diseased piglets, generated only one additional band in SDS-PAGE in accordance with a specific IgM cleavage product .
Complement activation leads to formation of C3 convertases (C3Bb or C4b2a) cleaving C3 into the anaphylatoxin C3a and the most important opsonin C3b. In an experimental mouse model the complement system proved to be crucial for protection against morbidity and mortality caused by intranasal S. suis infection, as recently demonstrated by our group using C3−/− mice . Thus, evasion of complement activation is essential for the survival of S. suis in its host and several factors involved in complement evasion have been identified in S. suis. Sialic acid moieties of the capsule of serotype 2 strains  might interfere with the activation of the alternative complement cascade by increasing the affinity constant of C3b to the complement inhibitor factor H [9,10]. Accordingly, deposition of C3b is increased on the bacterial surface of an unencapsulated mutant . Furthermore, two factor H binding proteins (Fhb and SSU0186) both homologous to PspC (Pneumococcal surface protein C) of S. pneumoniae have been identified in S. suis [11,12]. FhB was shown to contribute to virulence in experimental infections of piglets and to survival in human blood ex vivo.
The classical complement pathway is activated by immunoglobulins, in particular IgM, and some other host proteins, e. g. choline-binding protein, recognizing bacterial surface structures . Binding of the IgM pentamer to surfaces of pathogens leads to activation of the classical complement (c) cascade, as IgM, including porcine IgM, contains a C1q binding motif [14,15]. The results of this study showed that the cleavage site of Ide Ssuis in porcine IgM is located between the C1q-binding motif and the antigen recognizing part. Thus, we investigated whether IgM protease activity represents a novel complement evasion mechanism protecting the pathogen against classical complement activation.
S. suis strain 10 is a virulent serotype 2 strain that has previously been used for experimental infections of piglets and for generation of isogenic mutants [16-19]. It expresses the virulence-associated muramidase-released protein (MRP), the extracellular factor and suilysin . The capsule deficient isogenic mutant 10cpsΔEF is attenuated in virulence  and shows increased deposition of C3 antigen on its bacterial surface in murine serum . Streptococci were grown on Columbia blood agar plates or in BactoTM Todd Hewitt broth (THB). Escherichia (E.) coli strains were cultured in Luria-Bertani (LB) medium. If appropriate, antibiotics were added at the following concentrations: ampicillin, 100 μg/mL for E. coli; chloramphenicol, 3.5 μg/mL for S. suis, 8 μg/mL for E. coli; spectinomycin 100 μg/mL for S. suis.
Standard DNA manipulations were performed as described . Oligonucleotide primers were designed based on the sequence of SSU0496 in the genome of S. suis P1/7 . Chromosomal DNA of strain 10 served as template in all PCRs conducted for generation of inserts. DNA fragments were amplified with Phusion polymerase (Promega, Mannheim, Germany).
The mutant 10Δide Ssuis and its complemented strain 10Δide Ssuis pGA14ideSsuis were described previously . In frame deletion mutants expressing either the N-terminal part homologous to IdeS (10Δide Ssuis _C-terminus) or the large C-terminal part (10Δide Ssuis _homologue) were generated within this work using the thermosensitive plasmids pSET5Δide Ssuis _C and pSET5Δide Ssuis _h, respectively, to mutagenize S. suis strain 10. The following amplicons were generated with the indicated oligonucleotide primers to generate pSET5Δide Ssuis _C: a 619 bp ide Ssuis 5′-fragment amplified with ide Ssuis delCforPstI and ide Ssuis delCrevBamHI and a 608 bp ide Ssuis 3′-fragment generated with ide Ssuis delCforBamHI and ide Ssuis delCrevEcoRI (Additional file 1). Both fragments were cut with the restriction enzymes indicated in the name of the primers and inserted into the corresponding sites of pSET5. For the construction of pSET5Δide Ssuis _h a 614 bp 5′-ide Ssuis amplification product was generated with the primer pair preProIde Ssuis PstI plus postSSide Ssuis BamHI and a 621 bp 3′-ide Ssuis amplification product with the primer pair Ide Ssuis delh_for_BamHI and Ide Ssuis delh_rev_SacI (Additional file 1). Both fragments were cut with the indicated restriction enzymes and inserted into the corresponding sites in vector pSET5. Restriction analysis and sequencing was performed with pSET5Δide Ssuis _h and pSET5Δide Ssuis _C to verify both constructs.
The allelic exchanges for generation of 10Δide Ssuis _homologue and 10Δide Ssuis _C-terminus were performed essentially as described previously . The deletion of the genes was confirmed by PCR and Southern Blot analysis, which included four different probes for each mutant strain.
In frame deletion mutagenesis of ide Ssuis was conducted in the unencapsulated S. suis strain 10cpsΔEF with the thermosensitive plasmid pSET5Δide Ssuis constructed in our previous study . The unencapsulated double mutant 10cpsΔEFΔide Ssuis was confirmed by comprehensive Southern blot analysis using 4 different probes and two different digestions of DNA (HincII and BamHI).
The expression and the purification of the different recombinant Ide Ssuis constructs, MRP and the fibronectin-and fibrinogen-binding protein of S. suis (FBPS) were performed as previously described .
For αIgM Western blot analysis samples were prepared with reducing or non-reducing sample buffer and separated in 6% or 10% separating gels. For the detection of the Ig light chain the samples were prepared with reducing sample buffer and separated in a 12% separating gel. Western blot analysis was conducted as previously described  with antibodies specified together with the final dilution in Additional file 2. Polyclonal antisera were raised against Ide Ssuis , Ide Ssuis _homologue and Ide Ssuis _C-terminus in rabbits within our previous study .
The cleavage site of Ide Ssuis in IgM was determined through N-terminal sequencing after Edman degradation of a cleavage product. For this, recombinant Ide Ssuis in a concentration of 0.07 mg/mL was incubated with 0.68 mg/mL purified porcine IgM at 37 °C for 3 h on a rotator. The proteins were then separated under reducing conditions in a 10% separating and 4% stacking gel. The cleavage products were transferred to PVDF-membranes (Merck Millipore, Schwalbach, Germany) and either visualized in an αIgM Western blot or cut out for sequencing via N-terminal Edman degradation performed on an Applied Biosystems Procise Protein Sequencer 494C with reagents supplied by the manufacturer (Life Technologies, Darmstadt, Germany).
A hemolysis assay was established to investigate whether Ide Ssuis activity modulates the complement-dependent hemolysis caused by porcine Ig raised against ovine erythrocytes. For generation of sera containing these specific antibodies (αEry sera), two piglets were immunized with purified ovine erythrocytes and 10% Emulsigen (MVP Laboratories Inc., Omaha, NE). Generation of hyperimmune sera in pigs in our institute is registered under 12A226 at the Lower Saxonian State Office for Consumer Protection and Food Safety.
Sera were drawn from these piglets prior and post prime as well as post booster vaccination as indicated. Ovine erythrocytes from defibrinated blood were washed three times with 0.9% sodium chloride solution and were finally diluted to a 2% erythrocyte suspension. Hemolysis experiments were conducted with 1:20 final dilutions of porcine sera. To confirm that the hemolytic activity of the αEry sera depended on complement activity, sera were either inactivated by heat treatment (30 min 56 °C) or by addition of 10 mM EDTA (30 min 25 °C). The classical pathway was specifically inhibited by incubation of the serum with 10 mM EGTA and 15 mM MgCl2 (30 min 25 °C).
For functional analysis of Ide Ssuis , 1.8 μg recombinant protein of either Ide Ssuis or a truncated derivative was incubated with 100 μL of a 1:10 dilution of porcine serum (pre immune or αEry sera) for 1.5 h on a rotator at 37 °C. Erythrocytes were mixed with treated serum (each 100 μL) and incubated for 30 min at 37 °C on a shaker. As control the erythrocytes were incubated in water (defined as complete lysis of erythrocytes) or in a 0.9% sodium chloride solution. Unlysed red blood cells were pelleted by centrifugation (1000 × g for 5 min). One hundred μL of the supernatant was transferred into a 96-well flat bottom microplate and the absorbance was measured at 405 nm. For the inhibition assay, 5.2 μg recombinant protein was incubated with 0.4 M iodoacetamide (or as a control PBS) for 30 min. Unbound iodoacetamide was afterwards removed using Amicon Ultra 0.5 mL centrifugal filters with a 10 kDa cut off (Merck Millipore, Schwalbach, Germany). Subsequently, the iodoacetamide-inactivated Ide Ssuis contructs and its controls were investigated for modulation of hemolysis caused by αEry sera as described above but erythrocytes and serum were incubated for 1 h.
Porcine sera drawn before and after immunization with ovine erythrocytes (see above) were inactivated by heat treatment (30 min 56 °C). A 2% erythrocyte suspension generated from EDTA-blood was incubated with inactivated pre and post immune serum. Inactivated serum was pretreated with different rIde Ssuis constructs and as control with rMRP as described above to investigate a putative modulation of IgM and IgG binding to erythrocytes. After incubation with porcine sera, erythrocytes were centrifuged, resuspended in 5% goat serum (for the detection of IgM) or 5% rabbit serum (for the detection of IgG) and incubated on a rotator at 8 °C for 1 h. Erythrocytes were washed with PBS and incubated in a 1:250 dilution of a mouse anti-porcine IgM antibody (Serotec, Puchheim, Germany) or in a 1:10 000 dilution of a goat anti-porcine IgG antibody (Serotec) for 1 h at 8 °C. After washing of erythrocytes samples were incubated in a 1:500 dilution of a phycoerythrin-labelled goat anti-mouse IgG antibody (Bio Legend, Fell, Germany) or in a 1:1000 dilution of a Alexa fluor 488–labelled chicken anti-goat IgG antibody (Life Technologies, Darmstadt, Germany), respectively. Erythrocytes were analysed using a BD AccuriTM C6 (Becton Dickinson, Heidelberg, Germany) flow cytometer. For each determination 10 000 events were acquired and analysis of erythrocytes was carried out by dot plot analysis.
For opsonization of S. suis with C3b/C3i, 150 μL serum drawn after bacterin prime-vaccination or hyperimmune serum raised against S. suis serotype 2 was added to 75 μL of a culture grown to an OD600 of 0.8. After 1 h of incubation at 37 °C under rotation, bacteria were centrifuged, washed with PBS and incubated with a polyclonal FITC-labeled rabbit anti-human C3c antibody (Dako, Eching, Germany) (1:150 diluted in PBS) for 1 h at 8 °C. For opsonization of S. suis 10cpsΔEF and 10cpsΔEFΔideSsuis, 75 μL serum of colostrum-deprived piglets (SCDP) with or without the addition of purified porcine IgM (0.14 mg/mL) was added to 75 μL of a culture grown to an OD600 of 0.8. Porcine IgM were purified as described before (8). Bacteria and serum were incubated for 30 min at 37 °C and labelled with an antibody directed against C3 as described above. Fluorescent bacteria were analysed after washing with PBS and inactivation with 0.375% formaldehyde in flow cytometry as described previously .
To deplete serum of complement components, serum was pretreated with zymosan as decribed  with the following modifications. A 225 μL aliquot of a zymosan A (Sigma, Taufkirchen, Germany) stock solution (15 mg zymosan A resuspended in 1 mL of a 14 mM sodium chloride solution) was incubated for 30 min at 100 °C. The suspension was centrifuged at 16 000 × g for 5 min and the pellet was resuspended in 150 μL of porcine serum, incubated for 30 min at 25 °C and centrifuged at 16 000 × g for 5 min. Treatment of bacteria with this supernatant was compared to treatment of bacteria with untreated serum to access the effect of complement depletion by zymosan. All three complement pathways were blocked with 10 mM EDTA (30 min 25 °C) or heat inactivation (30 min 56 °C). To inhibit only the classical complement pathway, sera were incubated with 10 mM EGTA and 15 mM MgCl2 for 30 min at 25 °C. The differently treated serum samples were incubated with the bacteria which were subsequently analyzed for deposition of C3 antigen as described above.
Opsonophagocytic killing in the presence of 20% (v/v) porcine serum was assessed essentially as described . Porcine neutrophils were purified from freshly drawn blood as outlined previously . To obtain a multiplicity of infection of 0.03, 1.5 × 105 bacteria were added to 400 μL of a neutrophil suspension in RPMI containing 5 × 106 neutrophils and 100 μL serum. The samples were incubated for 1 h at 37 °C on a rotator. Samples incubated with porcine αS. suis serotype 2 hyperimmune serum and serum of colostrum-deprived piglets were included as positive and negative control, respectively. The survival factor as defined by the ratio of colony forming units (CFU) at t = 60 min to the respective value at t = 0 min was determined for each strain. The ratio of the survival factors of 10Δide Ssuis and wt was calculated to assess attenuation of the 10Δide Ssuis mutant.
Survival of S. suis in porcine blood was determined as described in a previous study . Briefly, 500 μL of heparinized blood (16 I. U. heparin/mL) were infected with 1.5 × 105 CFU using stocks of frozen bacteria with 15% glycerol after thawing. The blood was incubated for 2 h at 37 °C on a rotator. Bactericidal assays were conducted with blood drawn from 5 to 7 week old piglets 6 to 14 days after prime vaccination with a S. suis serotype 2 bacterin. These piglets were not included in the experimental infection experiment.
German Landrace piglets (n = 25) free of sly + mrp + epf + cps2+ strains were infected experimentally either with strain 10 (n = 9) or strain 10Δide Ssuis (n = 8) or 10Δide Ssuis _homologue (n = 8). Piglets were cared for in accordance with the principles outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes . The animal experiment of this study was approved by the Committee on Animal Experiments of the Lower Saxonian State Office for Consumer Protection and Food Safety (permit no. 33.9-42502-04-12/0965).
All piglets were prime-vaccinated at an age of 5–6 weeks with a bacterin generated with S. suis strain 10 grown overnight and inactivated in 0.2% formaldehyde. Emulsigen was added as adjuvant (20% [vol/vol]). Each immunization dose contained approximately 109 bacteria.
At an age of 7 to 8 weeks piglets were challenged 12 days after prime vaccination. Piglets were intranasally infected after predisposition through intranasal treatment with 1% acetic acid as described previously . Criteria for morbidity were fever (≥40.2 °C) or specific clinical signs such as convulsions or severe lameness. In the case of high fever (≥40.5 °C), apathy and anorexia persisting over 36 h as well as in all cases of clinical signs of acute polyarthritis or severe meningitis animals were euthanized for reasons of animal welfare. All surviving piglets were sacrificed 15 days post infection (dpi).
After euthanasia every animal went through the same procedure of necropsy including predefined collection of samples for histological and bacteriological investigations. Fibrinous-suppurative inflammations were scored in blinded experiments as described previously . To allow comparison of groups the sum of the highest scores of each animal for any of the investigated organs was divided by the number of animals (ω = Σscoremax/nanimals). Isolation of the challenge strains was confirmed in a PCR for detection of epf and cps2  and in ide Ssuis -specific PCRs using oligonucleotide primers specified in Additional file 1.
MRP, used as antigen for the IgG ELISA, is a dominant immunogen of this S. suis pathotype [17,23,27]. The detection of IgG titers against MRP was performed as described . For the measurement of αS. suis IgM or IgG antibody titers Maxisorb® plates (Nunc, Rochester, NY) were coated with 1 × 107 inactivated S. suis wt bacteria/well. Every sample and the controls were measured in a duplicate series of four (reference serum: seven) twofold dilutions in PBST starting with a dilution of 1:50. For the detection of S. suis specific IgM antibodies the plates were incubated with a dilution of 1:10 000 of a POD-conjugated goat anti-porcine IgM antibody (Thermo Scientific, Schwerte, Germany, catalog number PA1-84625) for 1 h at 37 °C. Blocking, washing and development of ELISA plates as well as calculation of ELISA units was conducted as previously described . Data were only considered if they met the following criteria: a deviation of duplicates of no more than 22%, a slope of the linear portion of the reference standard curve between 0.8 and 1.2, a correlation coefficient between 0.9 and 1.0, and controls within established ranges.
Experiments were performed at least three times and if not stated otherwise one-way analysis of variance (ANOVA) using Dunnetts adjustment or Tukeys multiple comparison test was used. ELISA-values were compared using the Mann–Whitney U-Test. Statistical analysis of Kaplan-Meier diagrams was conducted with the log-rank test. Means and standard deviation of the results are shown. Probabilities lower than 0.05 were considered significant (p < 0.05 *, p < 0.01 ** and p < 0.001 ***).
Cleavage of IgM by Ide Ssuis was previously identified and characterized in Western blot analysis under non-reducing conditions . In this study we used a different αIgM antibody recognizing only the reduced heavy chain of IgM to detect IgM cleavage products after Ide Ssuis incubation (Figure 1). This allowed us to successfully determine the N-terminal sequence of the 32 kDa cleavage product as SPITVFAIAP via Edman sequencing (Figure 1). Based on the N-terminal sequence, Ide Ssuis cleaves the heavy chain of IgM at the N-terminus of the C3 domain. In accordance with this result, reducing αIgM Western blot analysis revealed two cleavage products of 41 kDa and 32 kDa, which putatively included V1-C1-C2 and C3-C4 domains of the heavy IgM chain, respectively (Noteworthy, this αIgM antibody does not recognize the light chain of IgM).
Cysteines involved in disulphide bonds are conserved between human and porcine IgM.
Ide Ssuis cleaves the heavy chain of porcine IgM. (A) αIg light chain and αIgM Western blot analysis of purified porcine IgM after incubation +/− rIde Ssuis . Samples were separated in an SDS-gel under reducing conditions. The marker bands are shown on the left side (sizes in kDa). (B) The IgM-cleavage product highlighted in red was characterized by N-terminal Edman-sequencing. The identified sequence is shown in red and corresponds to the constant domain 3 of the IgM heavy chain. The putative C1q binding motif is marked in blue. Domains C2 and C4 are underlined. The obtained sequence was taken directly from the NCBI database. (C) Illustration of an IgM-monomer with the indicated cleavage site and the location of the putative C1q-binding motif.
Assuming that constitution of disulfide bonds is also conserved between the two species, the cysteines of C1 and C2 domains of porcine IgM should form intradomain and interchain disulphide bonds but not link monomers to the multimer. The Western blot in Figure 1 and further analysis with antibodies recognizing unreduced IgM  and the light chain of IgM only (Figure 1A), suggested that IgM is cleaved by Ide Ssuis only at the indicated site. IgM bound to the bacterial surface is also cleaved by Ide Ssuis and results in release of Fcμ cleavage products , which most likely include the C3 and C4 domain of the heavy chain of porcine IgM. This cleavage pattern is likely to affect IgM effector functions, in particular reduced activation of the classical complement pathway, as the C3 domain includes the C1q binding motif of porcine IgM [28,29].
The classical complement activation pathway can be studied in hemolysis assays using sera containing antibodies directed against erythrocytes. We investigated the impact of Ide Ssuis on complement activation in a hemolysis assay including sera drawn from piglets vaccinated with erythrocytes (αEry sera). In accordance with complement activation, treatment of αEry sera with heat, EDTA or EGTA plus MgCl2 completely abolished the hemolytic activity of the αEry sera (Figure 2A). For functional analysis of Ide Ssuis , αEry sera drawn after prime and booster vaccination were treated with different rIde Ssuis constructs (Figure 2B) prior incubation with erythrocytes. Incubation of the post-prime αEry serum with rIde Ssuis and rIde Ssuis _homologue (the domain containing the IgM protease), almost completely abolished this hemolysis (Figure 2C). Noteworthy, treatment of the post-prime αEry serum with two recombinant control proteins (MRP and FBPS), did not result in abrogation of hemolysis (Figure 2C). Interestingly, treatment of αEry sera with proteolytic inactive construct rIde Ssuis _C_terminus led also to a significant reduction of hemolysis indicating a separate role of the C-terminus in complement evasion. However, significant differences between inhibition of complement activation through αEry sera drawn after prime and booster vaccination were only observed for the Ide Ssuis constructs with IgM protease activity (Figure 2C).
Hemolysis caused by the classical complement activation pathway is abrogated by Ide Ssuis in dependence of the protease activity. (A) Purified sheep erythrocytes were incubated with water (defined as 100% hemolysis), physiological sodium chloride solution (NaCl), sera of a piglet drawn before (pre vaccination serum) and 7 days after prime vaccination (post vaccination αEry serum) with ovine erythrocytes. The αEry serum was heat inactivated or treated with EDTA to access the impact of complement activation. To specifically inhibit the classical complement pathway 10 mM EGTA plus 15 mM MgCl2 was used. (B) Illustration of rIde Ssuis and its truncated derivatives. The amino acids of Ide Ssuis included in these constructs are superscribed. The region homologous to IdeS is shaded. (C) Complement dependent hemolysis is reduced by pretreatment of the indicated different αEry sera with rIde Ssuis , rIde Ssuis _homologue (rIdeSsuis_h) and rIde Ssuis _C_terminus (rIdeSsuis_C) but not with rMRP and rFBPS. (D) Proteolytic activity of rIde Ssuis and rIde Ssuis _homologue is crucial for complement inhibition. The Ide Ssuis constructs were incubated with the cysteine-protease inhibitor iodoacetamide prior to incubation with the αEry sera as indicated. The final dilutions of porcine sera in the hemolysis assay were 1:20 in all cases. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).
The different recombinant Ide Ssuis constructs were treated with the protease inhibitor iodoacetamide prior to incubation with αEry serum to assess the impact of proteolytic activity on complement inhibition. Preincubation of rIde Ssuis and rIde Ssuis _homologue with iodoacetamide completely abrogated the complement inhibiting activity of these proteins (Figure 2D).
Furthermore, flow cytometry analysis was conducted with erythrocytes after incubation with inactivated post prime and post booster αEry sera to differentiate binding of specific IgM and IgG in these sera. Both sera contained erythrocyte-specific IgG and IgM in contrast to the pre immune serum. Incubation with the post prime serum led to significantly higher percentage of IgM-labelled erythrocytes and respective mean fluorescence intensity (MFI) in comparison to the post booster serum. Vice versa, IgG staining on erythrocytes resulted in a much higher MFI after incubation in post booster serum (Figures 3A and B). We investigated modulation of IgM and IgG antigen binding to erythrocytes by treatment of the post prime αEry serum with the different recombinant Ide Ssuis constructs. Treatment of this serum with proteolytic active rIde Ssuis and rIde Ssuis _homologue led to a significant reduction of the percentage of erythrocytes labelled with IgM and the respective MFI (Figures 3C and D) in contrast to the treatment with the non-proteolytic constructs. Binding of IgG to the erythrocytes was not modulated by incubation of the αEry serum with any of the recombinant Ide Ssuis constructs.
The proteolytic rIde Ssuis constructs modulate IgM binding to the surface of erythrocytes after incubation in αEry sera. (A, B) Flow cytometry analysis of IgM and IgG binding to ovine erythrocytes after incubation with porcine sera drawn pre, post prime or post booster vaccination with ovine erythrocytes (generation of αEry sera). (C, D) Binding of specific IgM antigen to the surface of ovine erythrocytes is significantly reduced after pretreatment of a post prime αEry serum with rIde Ssuis and rIde Ssuis _homologue. The post prime αEry serum was preincubated with the indicated Ide Ssuis constructs and as a control with MRP. (A, C) The percentage of erythrocytes labelled with IgM or IgG and (B, D) the mean fluorescence intensities are shown. αEry sera were inactivated in these assays to allow flow cytometric analysis of erythrocytes. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (***p < 0.001) except for differences between pre and post vaccination sera.
In conclusion, Ide Ssuis interferes with the classical complement activation pathway. The results of the hemolysis assay suggest that Ide Ssuis interferes with complement activation by two mechanisms, firstly, by its IgM protease activity and, secondly, by some yet unknown function of the large non-proteolytic C-terminal domain.
We investigated deposition of C3 on the surface of opsonized S. suis strains by flow cytometry to further investigate the hypothesis that Ide Ssuis is involved in complement evasion. After opsonization of S. suis wt and 10Δide Ssuis with different porcine sera with moderate to high specific IgM titers the percentage of bacteria with stained C3 antigen (most likely C3b/C3i) was slightly increased in the mutant 10Δide Ssuis (Additional file 3). Specifically, 39.2% of wt (SD = 11.0%) and 42.8% of 10Δide Ssuis (SD = 10.2%) bacteria were C3-labelled after opsonization in sera of bacterin-primed piglets (Additional file 3). The sera of these bacterin-primed piglets had moderate to high titers of αS. suis IgM (34 – 103 ELISA units) and αS. suis IgG (69 – 161 ELISA units) but low αMRP titers (below 15 ELISA units). After addition of EGTA and MgCl2 to these sera only 7.1% (SD = 4.8%) and 7.7% (SD = 6.8%) of wt and 10Δide Ssuis bacteria, respectively, were labelled with the αC3 antibody indicating that complement is mainly activated by the classical activation pathway during early adaptive immune responses (Additional file 3).
We hypothesized that activation of the classical complement pathway in this assay was determined by specific IgM and IgG and that redundant complement evasion mechanisms in S. suis serotype 2 might limit detection of phenotypic differences between wt and 10Δide Ssuis . Thus, we deleted ide Ssuis in an unencapsulated isogenic strain (10cpsΔEFΔide Ssuis ) to avoid complement inhibition through the sialylated capsule of S. suis serotype 2. Furthermore, effects of specific IgG were excluded using serum from colostrum-deprived piglets (SCDP). As shown in Figure 4A approximately 10% of either 10cpsΔEF or 10cpsΔEFΔide Ssuis bacteria were stained with C3 after opsonization with SCDP. Importantly, the percentages of C3-stained 10cpsΔEF increased to about 20% after addition of purified porcine IgM to SCDP prior to opsonization, but fourfold (to 40%) for 10cpsΔEFΔide Ssuis bacteria lacking IgM protease activity (Figure 4). Differences in C3 deposition between 10cpsΔEF and 10cpsΔEFΔide Ssuis were highly significant for the percentage of labelled bacteria and the MFI (Figure 4). Addition of the classical complement pathway inhibitor (EGTA plus MgCl2) reduced the percentage of C3 labelled bacteria to 4.7% (10cpsΔEF) and 4.9% (10cpsΔEFΔide Ssuis ) and diminished the phenotype of the double mutant. These results confirm that C3 deposition on the surface of S. suis might be determined by IgM-mediated activation of the classical complement pathway and show that S. suis reduces this IgM-mediated C3 deposition by expression of Ide Ssuis .
Expression of Ide Ssuis reduces C3 deposition on the surface of an unencapsulated mutant triggered by IgM-mediated activation of the classical complement pathway. C3 antigen bound to the surface of the unencapsulated mutant 10cpsΔEF and the double mutant 10cpsΔEFΔide Ssuis was detected through flow cytometry analysis. Bacteria were opsonized with sera from colostrum-deprived piglets (SCDP) or SCDP spiked with purified porcine IgM (SCDP plus IgM). To inactivate the classical complement activation pathway SCDP plus IgM was pretreated with EGTA MgCl2. As control non-opsonized bacteria (incubated in PBS) were incubated with the C3 specific antibody. (A) The percentage of bacteria with antibody-labelled C3 and (B) the mean fluorescence intensity of the samples are shown. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
As rIde Ssuis interfered with complement activation using sera with specific antibodies, we hypothesized that expression of the IgM protease Ide Ssuis contributes to survival in opsonophagocytosis assays including a porcine serum with specific IgM and comparatively low specific IgG titers (αS. suis IgM: 29.2 ELISA-units and for comparison: αMRP IgG: 12.8 ELISA-units). Phenotypic analysis of S. suis was conducted in this study using the mutant 10Δide Ssuis  and two new in frame deletion mutants expressing truncated Ide Ssuis constructs. These new mutants, designated 10Δide Ssuis _C-terminus and 10Δide Ssuis _homologue, expressed the N-terminal part homologous to IdeS and the large C-terminal part lacking homologies, respectively (Additional file 4). Noteworthy, 10Δide Ssuis _C-terminus released IgM protease activity in the supernatant in contrast to 10Δide Ssuis and 10Δide Ssuis _homologue (Additional file 4). As shown in Figure 5A 10Δide Ssuis and 10Δide Ssuis _homologue had a significant lower survival factor compared to the wt strain and the survival factor for 10Δide Ssuis _C-terminus was also found to be lower compared to the wt strain. The extent of attenuation of 10Δide Ssuis was significantly lower in opsonophagocytosis assays including serum from a colostrum-deprived piglet in comparison to assays including specific IgM. Inhibition of complement reduced the attenuation of the mutant 10Δide Ssuis significantly (Figure 5B).
Ide Ssuis promotes survival in opsonophagocytosis assays including purified porcine neutrophilic granulocytes and serum with specific IgM titers. (A) Survival of strain 10 (wt), 10Δide Ssuis (Δ), 10Δide Ssuis _homologue (Δ_h) and 10Δide Ssuis _C-terminus (Δ_C) in opsonophagocytosis assays with serum from a piglet with specific IgM. (B) Attenuation of 10Δide Ssuis in opsonophagocytosis assays depends on active complement and adaptive immunity. The ratios of the survival factors of 10Δide Ssuis to the respective survival factors of wt are shown. A ratio of 1 is depicted by the horizontal line and refers to a lack of attenuation. To access the impact of complement, the post immune serum with specific IgM used for experiments shown in (A) was heat-inactivated or treated with zymosan. Samples with active and heat-inactivated serum from a colostrum-deprived pig were included to investigate whether this Ide Ssuis -mediated phenotype depends on adaptive immunity. Bars and error bars represent mean values and standard deviations, respectively. Significant differences between strains and ratios of survival factors in (A) and (B), respectively, are indicated (* p < 0.05, ** p < 0.01).
Based on the in vitro results we considered Ide Ssuis to be a putative virulence factor of S. suis in piglets with high titers of specific IgM. Thus, we conducted experimental infection of piglets prime vaccinated with a bacterin. Immunological screening of these piglets confirmed that these piglets had high specific IgM titers and low IgG titers against MRP, a main immunogen of this invasive S. suis pathotype (Additional file 5). We infected piglets with the wt and the 10Δide Ssuis mutant as well as with the partial mutant 10Δide Ssuis _homologue, which expressed only the C-terminus and showed no IgM proteolysis (Additional file 4). The complemented mutant was not included in the experimental infection because it showed attenuation in growth in medium (unpublished results). Sixty three percent of the piglets infected with the mutant 10Δide Ssuis survived this experiment, whereas only 33% did so in the wt infected group (p = 0.125) (Figure 6A). Furthermore, 50% and 11% of 10Δide Ssuis and wt infected piglets, respectively, were free of clinical signs throughout the observation period (p = 0.076; Figure 6B). Furthermore, 10Δide Ssuis infected piglets had a lower pathohistological score (ω = 2.2) in comparison to wt infected animals (ω = 3.7; Table 1). In general, detection of fibrinosuppurative lesions was associated with detection of the infection strain. The mutant 10Δide Ssuis was not detectable in any inner organ in 5 of 8 infected piglets (Table 2). However, the group infected with the deletion mutant 10Δide Ssuis _homologue showed mortality and morbidity as well as a high rate of infection of inner organs very similar to the wt infected group (Figure 6; Table 2).
Mortality (A) and morbidity (B) of prime-vaccinated growing piglets intranasally challenged with the indicated S. suis strains. Growing piglets were infected with wild type strain 10 (wt), 10Δide Ssuis (Δ) and 10Δide Ssuis _homologue (Δ_h) 12 days after prime vaccination with a homologous S. suis serotype 2 bacterin. A piglet was determined as morbid in the case of elevated body temperature (≥40.2 °C) or specific clinical signs (signs of central nervous dysfunction or severe lameness). Statistical analysis of the Kaplan-Meier diagrams was conducted with the log-rank test (all p-values are shown below the diagrams).
aInfection strains were strain 10 (wt), 10Δide Ssuis (Δ) and 10Δide Ssuis _homologue (Δ_h).
bNeutrophilic accumulation of the splenic red pulp.
cScoring of 4 and 5 indicates moderate to severe diffuse or multifocal fibrinosuppurative inflammations.
dScoring of 2 and 3 indicates mild focal fibrinosuppurative inflammation.
eIndividual single perivascular neutrophils received a score of 1.
aInfection strains were strain 10 (wt), 10Δide Ssuis (Δ) and 10Δide Ssuis _homologue (Δ_h). Identification was conducted through PCR as described in Materials and methods.
bPiglets with isolates of the challenge strain exclusively from the tonsil were not considered.
cOne cranial lobe was investigated.
dPleural, peritoneal or pericardial cavity.
fPunctures of both tarsal and carpal joints were investigated in each animal. In cases of lameness additional joint punctures of the respective limb were screened.
In summary, the results of the experimental infection suggested an attenuation of the mutant 10Δide Ssuis in prime-vaccinated growing piglets with high titers of specific IgM.
As bacteremia is considered to be a critical step in the pathogenesis of invasive S. suis diseases, we further investigated survival of the different ide Ssuis mutants and the wt in porcine blood with high IgM titers ex vivo. Thus, we evaluated Ide Ssuis -dependent survival in blood from bacterin prime-vaccinated piglets. These piglets had significantly higher IgM titers against S. suis than unvaccinated weaning piglets investigated for comparison (p < 0.01; Additional file 6). As shown in Figure 7, the two mutants deficient in IgM proteolysis (10Δide Ssuis and 10Δide Ssuis _homologue) are significantly attenuated in survival in blood drawn from piglets prime-vaccinated with a bacterin. In contrast, the mutant expressing the truncated N-terminal domain with IgM protease activity (10Δide Ssuis _C-terminus) was not attenuated in growth in porcine blood ex vivo.
The mutants 10Δ ide Ssuis (Δ) and 10Δ ide Ssuis _homologue (Δ_h) are attenuated in survival in porcine blood ex vivo. The blood was drawn from 7 different growing piglets with an age of 5–7 weeks 6–14 days after prime vaccination with a S. suis bacterin. The mutant 10Δide Ssuis _C-terminus (Δ_C) not deficient in IgM proteolysis was also included. The bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (** p < 0.01; *** p < 0.001).
In conclusion, expression of the IgM protease Ide Ssuis promotes increased survival of S. suis in porcine blood ex vivo, at least in the presence of specific IgM.
The IgM pentamer is a very important activator of the classical complement pathway. It has been estimated that the efficiency of one pentameric IgM molecule to activate complement is equivalent to the respective efficiency of 1000 IgG molecules . As cleavage of IgM by Ide Ssuis occurs at a site located between the antigen-recognizing part and the Fc-part containing the putative C1q binding motif, it was reasonable to hypothesize that IgM cleavage by Ide Ssuis is an important complement evasion mechanism of S. suis. In this study we obtained in vitro data supporting the hypothesis that Ide Ssuis is involved in complement evasion: (i) different recombinant Ide Ssuis constructs abolished the hemolysis induced by activation of the classical complement pathway in serum with specific IgM directed against erythrocytes; (ii) attenuation of the mutant 10Δide Ssuis in opsonophagocytosis assays was complement-dependent; (iii) IgM-triggered deposition of C3 on the bacterial surface is reduced by Ide Ssuis expression and (iv) attenuation of 10Δide Ssuis in survival in porcine blood ex vivo was observed in blood from piglets with high specific IgM titers.
The hemolysis assays of this study showed that Ide Ssuis interferes substantially with activation of the classical complement pathway. Importantly, Ide Ssuis mediated inhibition of complement activation by erythrocyte specific IgM was abrogated by pretreatment of Ide Ssuis and Ide Ssuis _homologue with the protease inhibitor iodoacetamide. As IgM is the only known substrate of this protease  and inhibition of IgM proteolysis by iodoacetamide was confirmed in this assay, we conclude that IgM proteolysis is involved in interference of Ide Ssuis with complement activation. Accordingly, C3 deposition on 10cpsΔEFΔide Ssuis bacteria in serum from colostrum-deprived piglets spiked with specific IgM is significantly higher compared to C3 deposition on the Ide Ssuis expressing strain 10cpsΔEF.
Though IgM is much more powerful in activation of the classical complement pathway than IgG, immune evasion mechanism of bacteria counteracting the classical complement pathway have so far only been described for factors interacting with IgG. Specifically, protein H expressed by group A streptococci (GAS) reduces C3 deposition on IgG-coated beads and inhibits immune hemolysis of IgG-sensitized erythrocytes . Furthermore, protection against opsonophagocytic killing of GAS in the presence of specific IgG is mediated by M-proteins and M-like proteins acting as Fc-receptors  and by the IgG protease IdeS . In light of the different virulence factors counteracting IgG-mediated activation of the classical complement pathway, it is very much surprising that a bacterial evasion mechanism counteracting IgM-mediated complement activation has to the best of our knowledge not been described. The interference of Ide Ssuis with IgM-mediated complement activation is important for pathogenesis, since survival of S. suis in porcine blood of prime-vaccinated piglets is significantly increased by Ide Ssuis expression and clinical as well as pathological findings after experimental infection of respective piglets suggested attenuation of the isogenic mutant 10Δide Ssuis .
Deposition of C3b on the surface of the encapsulated S. suis serotype 2 strains was only slightly determined by Ide Ssuis expression under the chosen experimental conditions. The percentage of bacteria with detectable C3b deposition was below 16% after incubation of S. suis strains 10 and 10Δide Ssuis in serum of colostrum-deprived piglets supplemented with purified porcine IgM (results not shown) in contrast to the results shown for the unencapsulated mutants (Figure 4). This indicates in accordance with published results  that the capsule of serotype 2 is a main inhibitor of C3b deposition. Thus, we speculate that Ide Ssuis expression might be crucial for bacterial survival (i) during reduced capsule expression, (ii) in the presence of very high IgM titers against the polysaccharide capsule and (iii) in strains of serotypes that lack capsular sialic acid .
Two factor H binding proteins, have recently been identified in S. suis [12,13]. Factor H bound to the surface of S. suis serves as a cofactor for the factor-I mediated cleavage of C3b . Deletion of the gene fhb encoding one of the factor H binding proteins led to a significant increase in C3b/iC3b deposition after opsonization with human serum. Activation of human complement was elicited by S. suis mainly via the alternative pathway under the chosen experimental conditions. However, in this work we demonstrate that in growing piglets with high titers of specific IgM the percentage of bacteria with antibody-labelled C3 is mainly determined by the classical pathway. This is important, because piglets with early adaptive immune responses are often affected by S. suis diseases and development of vaccines eliciting protection during this early immune response stage would substantially improve animal health.
Cleavage of IgM might have important biological consequences in addition to prevention of C3b deposition on the bacterial surface. Based on the identified cleavage site in the IgM heavy chain, Ide Ssuis activity should lead to release of a pentameric Fc-molecule including only C3 and C4 domains of the heavy IgM chains. We speculate that the cleavage product detected above the 250 kDa marker lane in Western blot analysis under non-reducing conditions  constitutes this pentameric molecule. Future studies should consider whether this putative C3-C4-pentamer modulates functions of the immune system. It is known that ½Fc IgG fragments released upon cleavage of IgG by IdeS prime neutrophils to respond to a second stimulus with an enhanced rate of reactive oxygen species production . This might lead to activation of immune cells at sites remote from the pathogen. Similarly, the putative C3-C4 pentamer might also activate immune cells, e.g. by binding to the Fcμ receptor .
The results of the experimental infection suggested attenuation of the mutant 10Δide Ssuis . However, the mutant expressing only the C-terminus of Ide Ssuis (10Δide Ssuis _homologue) caused morbidity in prime-vaccinated piglets comparable to the wt and unlike the mutant 10Δide Ssuis , which suggests that IgM proteolysis as such was not crucial for the outcome of the animal experiment and that the C-terminus of Ide Ssuis carries out important, yet unknown functions. Accordingly, the recombinant truncated protein consisting only of the C-terminus of Ide Ssuis showed also a significant interference with complement activation in the hemolysis assay suggesting an additional function of Ide Ssuis in complement inhibition but IgM proteolysis. Interestingly, the interference of this non-proteolytic construct did not seem to depend on the ratio of erythrocyte-specific IgM and IgG titers, in contrast to the interference by the IgM protease domain (as estimated by comparative analysis of post prime and post booster αEry sera). Further studies are certainly needed to decipher further functions of Ide Ssuis in complement evasion and their role in host-pathogen interaction.
Host-pathogen interaction of S. suis was investigated during early adaptive immune responses in this study. Survival in porcine blood with high specific IgM titers is significantly determined by expression of the IgM protease Ide Ssuis . Accordingly, the in vitro results of this study demonstrate that Ide Ssuis abrogates activation of the classical complement system. As Ide Ssuis is expressed by all investigated S. suis strains , this unique virulence mechanism appears to be crucial for the evolutionary success of this pathogen.
We thank H. Smith (DLO-Lelystad, Netherlands) for S. suis strains 10 and 10cpsΔEF. Daisuke Takamatsu (National Institute of Animal Health, Japan) kindly provided the plasmid pSET5s. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany (SFB587), IDT Biologika GmbH and by the German Federal Ministry for Research and Education (BMBF) within the Helmholtz – CAS – Joint Research Group ZooStrep (HCJRG-116). UvPR is supported by the Carl Tryggers foundation, Sweden. We acknowledge support from the German Research Foundation (DFG) and Universität Leipzig within the program of Open Access Publishing.
Additional file 1: Sequences of oligonucleotide primers. Name, sequence and position of target sequence of primers used in this study.
Additional file 2: Antibodies used in Western blot analysis. Specificity, source, conjugation and used dilution of antibodies.
Additional file 3: Flow cytometry analysis of C3 antigen (C3b/C3i) bound to opsonized S. suis strain 10 (wt) and 10Δide Ssuis (Δ). Mean values and standard deviations (S. D.) for the percentage of C3-labelled bacteria and respective mean fluorescence intensity after opsonization of bacteria with sera including specific immunoglobulins as indicated.
Additional file 4: 10Δide Ssuis _ homologue (Δ_h) and 10Δide Ssuis _ C-terminus (Δ_C) release stabile fragments of Ide Ssuis into the supernatant in accordance with IgM cleavage activity in the case of Δ_C. (A) αIdeSsuis_C-terminus and αIdeSsuis_homologue Western blot analysis of culture supernatants of 10Δide Ssuis _homologue (Δ_h), 10Δide Ssuis _C-terminus (Δ_C), 10Δide Ssuis (Δ) and wild type strain 10 (wt). (B) 10Δide Ssuis _C-terminus but not 10Δide Ssuis _homologue exhibits IgM-cleaving activity. αIgM Western blot analysis of diluted porcine serum incubated with concentrated culture supernatants of 10Δide Ssuis _homologue (Δ _h), 10Δide Ssuis _C-terminus (Δ_C), 10Δide Ssuis (Δ) and wild type strain 10 (wt) or with PBS.
Additional file 5: Immunological analysis of piglets used in the experimental infection (see Figure 6 ). (A) α-S. suis serotype 2 (ST2) IgM, (B) α-ST2 IgG titers and (C) α-MRP IgG titers were determined in serum samples of piglets after prime vaccination with a S. suis ST2 bacterin (before experimental infection) and as a control in unvaccinated piglets. Significant differences are indicated. Mean values are shown by horizontal lines.
Additional file 6: Immunological analysis of piglets used for the bactericidal assay (see Figure 7 ). (A) α-S. suis serotype 2 (ST2) IgM and (B) α-MRP IgG titers were determined in serum samples of these 7 growing piglets (prime-vaccinated) and for comparison in unvaccinated piglets. Prime vaccination was conducted with a S. suis serotype 2 bacterin. Significant differences are indicated (** p < 0.01). Horizontal lines represent mean values.
JS conducted mutagenesis, all complement assays and phenotyping of the mutants in vitro. Furthermore, she supported experimental infections, conducted statistical analysis and participated in experimental design and drafting the manuscript. AB conducted histopathological analysis. Immunological analysis of the piglets was performed by LMH. BJK conducted N-terminal sequencing of the cleavage products. UvPR and PVW participated in study design and manuscript revision. CGB conceived of the study, designed experiments, conducted experimental infections and drafted the manuscript. All authors read and approved the final manuscript.


Mystery is surrounding the sudden closure of a shop on Lincoln High Street.
The shutters are down on Superfi on the upper end of the street, with no sign to say why, and stock is still visible inside the premises.
Superfi has stores all across the UK including Nottingham, Manchester, Birmingham, Derby and London, but Lincolnshire Live has called all and none have answered the phone.
According to What Hi-Fi, the company has told suppliers in an email it intends to enter into administration.
The email said: "We have filed notice of intention to appoint an administrator to the business on 5th February."
The chain brands itself as the most trusted supplier of hi-fi, home cinema, TV and multi-room equipment offering expert advice and low prices.
However, it appears that customers have been experiencing problems over the last couple of days.
On Twitter, Helen Warner said that she has been trying to get in touch with Superfi since Tuesday, February 5 but to no avail.
She tweeted: "@Superfionline why are no stores answering phone calls? #Nottingham #Lincoln #Manchester."
Another user tweeted: "So what's going on with Superfi? I have a lot on order but no answer from any number and the stores I checked were closed."
One concerned shopper contacted Lincolnshire Live and said: "I have been using Superfi on Lincoln High Street for quite some time.
"However, today when I went past the shutters were all down and the lights were off.
"I tried to call a number of stores but none of them answered.
"I come to Lincoln to use the shop and I knew the staff really well. It has really shocked me."
Lincolnshire Live has tried on numerous occasions to contact Superfi, but at the time of publication has received no answer.
The Financial Conduct Authority have said that they are looking into the issue.


- U2000-IP design( cooperate with customer to prepare the design solution, discussion and finalize it) for test and product environment.
- U2000-IP implementation on production environment(commissioning, integration with Huawei Node CX600-X8A).
- CCNP certification with Good IPV4/IPV6 knowledge about IP Core, NAT, Bras.
- Familiar with Huawei U2000-IP system.


In this adventure you will personify Khara, the daughter of a humble fisherman who is shipwrecked together with her father on a mysterious island: the mythical Atlantis.
Khara will discover that her origins tie her more to that land than she could have imagined and that incredible powers that allow her to control the water have always been within her.
A thrilling and exciting journey full of dangers in which you will help Khara to face her destiny and become a young heroine.
Como ya sabréis, el jueves pasado presentamos el prototipo a profesionales del sector y a la prensa, concretamente a HobbyIndustria, y parece que la cosa fue bien, porque recibimos un muy buen feedback.
El estudio español afincado en Madrid está preparando su primer trabajo titulado Khara para la plataforma PlayStation 4.
Somos un estudio indie de videojuegos formado por alumnos de los másters de game design, arte y programación de la U-tad, y estamos desarrollando nuestro primer videojuego nada más y nada menos que para PS4.


25 March 2019 – President of the South African Football Association (SAFA) Dr Danny Jordaan has congratulated Bafana Bafana for qualifying for the 2019 Africa Cup of Nations Tournament that will be played in Egypt in June.
This comes after the South African Senior Men's National Team registered a hard-fought 2-1 victory over a stubborn Libya on Sunday, 24 March 2018.
The match was played in Sfax, Tunisia.
Percy Tau's brace secured the win for Bafana Bafana, who finished the qualifiers unbeaten in six matches – and conceded only two goals.
Speaking to the media after watching the match on the big screen at the Nelson Mandela Square in Sandton, where he was joined by, among others, Minister of Sport and Recreation South Africa Thokozile Xasa, injured Bafana Bafana goalkeeper Itumeleng Khune, some Proteas and Springboks players, and some fans, Dr Jordaan hailed Bafana Bafana for their strong heart and mental attitude.
This was an important victory because you have to be part of the 24 team on the continent that will be at the tournament so that we can stand a chance to be in the top eight out of the 24, because if in June we agree that the World Cup in 2022 will be 48 teams, it means that Africa will have nine or ten teams, and therefore if we are in the top eight we will be topping the group for the World Cup qualification rounds. So it was important to qualify for that reason. We are in there now, so we must work hard to get into the top eight or nine, and then look at qualification for the World Cup, and we have to qualify for that World Cup. That will be the ultimate dream of Vision of 2022 – we have achieved everything: The U17 girls qualified for the World Cup in 2018, the U20 boys qualified for the World Cup in 2019, Banyana Banyana qualified for the World Cup in 2019, now Bafana Bafana have qualified for the AFCON. It is just the World Cup for the Senior Men that is left.
From a strategic point of view, for any country it must be important to qualify for such tournaments, but for the targets of AFCON we need the coach to come back, and give us the evaluation of the entire AFCON qualification. You must know that in our group we are the only team that is unbeaten – and when I told you this, you responded by telling me about realism. It is realistic that from the beginning to the end of these AFCON qualifiers, the only unbeaten team in the group is South Africa – that is a fact and a reality. So when he (Stuart Baxter) comes back, we have to look at why when it comes to Seychelles we score six goals and cannot score a single goal in the return match – that talks to consistency.


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Hydrogen? Just add water and sunlight
Hydrogen has been sold to the public as having the potential to be the ultra-clean fuel for the future's economy. What's less likely to be mentioned is that 96% of hydrogen is produced from natural gas, coal or other fossil fuels – producing it using renewable electricity is simply too expensive. To realise hydrogen's full potential, the world needs better means of splitting water into hydrogen and oxygen. Professor Kazunari Domen at the University of Tokyo/Shinshu University is performing research into how we can improve a method which generates hydrogen from only water and sunlight: photocatalysis.
Hydrogen has developed a name for itself as the ultra-clean fuel of the future. If you ran a car on hydrogen, only water would come out of its exhaust pipe.
But there's a problem. About 96% of the world's hydrogen is formed from fossil fuels, the majority of which is produced by the steam reforming of natural gas. Methane has its hydrogen atoms removed, forming hydrogen gas, and the atoms are replaced with oxygen – forming carbon dioxide which is a greenhouse gas. One tonne of hydrogen comes at the expense of creating 9 to 12 tonnes of carbon dioxide.
It's true that there are methods for producing hydrogen which don't rely on fossil fuels – electrolysis of water is perhaps the more famous hydrogen-production method. However, its energy consumption means that the resulting hydrogen comes at about double the cost of hydrogen from natural gas. As a result, only 4% of the world's hydrogen is produced by electrolysis.
Photocatalysts
If hydrogen is really going to be the low-carbon future fuel, or even a minor part of a green economy, we will need another method to produce hydrogen.
Photocatalysts are semiconductor materials that absorb light and catalyse reactions using the collected energy. Photocatalysts are beginning to receive research attention for their ability to convert water into hydrogen and oxygen using just sunlight.
angellodeco/Shutterstock.com
Photocatalysts used for water splitting are generally a fine powder. At a laboratory scale, the particulate photocatalyst is conventionally suspended in water so that the powder is distributed around the whole volume of water.
But will this system work at a larger scale? A suspended particulate reactor would require shallow water, but even with water 1 cm deep, the weight of water in the reactor would be 10 kg per square meter. This very thin layer of water would need continuous stirring to maintain the suspension, which is difficult over a large area. As a result, the more this technology is scaled, the less likely it would be to work.
Professor Kazunari Domen, at the University of Tokyo is researching solutions to scalability issues for these photocatalysts. His research focuses on a more scalable approach: 'fixed particulate photocatalysts'. His group constructed a reactor composed of two acrylic panels, with room between them for a thin layer of water to sit on top of a sheet of the photocatalyst deposited onto a sheet of glass. Rather than swilling around in the water, the photocatalysts are fixed in place on "photocatalyst sheets".
If hydrogen is really going to be the low-carbon future fuel… we will need scalable and low-cost methods to produce hydrogen without CO2 production.
In 2019, Prof Domen published details of his group's laboratory scale tests, initially of smaller 5-by-5 cm photocatalyst sheets under bright UV light illumination with a depth of 1 mm. The photocatalyst was 'aluminium-doped strontium-titanium oxide' (SrTiO3:Al), and is one of the most active photocatalysts for water splitting at ambient pressure using sunlight – good properties for real-world applications.
Scanning electron microscope images showing morphology of SrTiO3:Al photocatalysts with almost 100 % IQY.
With bench-scale testing confirming that the fixed panel photocatalyst method was viable – able to evolve hydrogen and oxygen at a reasonable rate, Domen and his team built several larger panel reactors with a total area of 1 m2, to demonstrate the scalability of fixed particulate sheets.
A panel with only 1mm of water was capable of rapid hydrogen and oxygen production without the need to stir or force convection through the reactor. But how efficiently? The key figure Domen is looking at is the solar-to-hydrogen energy conversion efficiency (STH).
The STH of the panels is around 0.4% under natural sunlight, which was comparable to the STH from laboratory-scale experiments under simulated sunlight. However, it is estimated that commercial photocatalytic solar hydrogen production would require an STH around 5-10% to be financially viable.
Tuning the photocatalyst
Domen's latest research, published in Nature, demonstrated methods to construct highly efficient photocatalyst particles using SrTiO3:Al as a typical example. The photocatalyst must have a high apparent quantum yield (AQY), a measure of the efficiency that photons which introduced into a reactor are used in the water splitting reaction. The AQY value is important because it is essential to improve the STH value – the solar energy to hydrogen conversion.
Detailed structure of a SrTiO3:Al photocatalyst particle with HER and OER co-catalysts.
Over the last few years, various refinements had improved the AQY of this photocatalyst to 69%. And finally AQY reached more than 95% at 350-360 nm, which is almost upper limit of AQY because it means nearly 100% of internal quantum yield (IQY). IQY is the efficiency of photon utilisation absorbed by photocatalyst itself.
The approach involves locating efficient co-catalysts like rhodium, chromium and cobalt at suitable position of a particulate photocatalyst using a manufacturing method called photodeposition.
These co-catalysts were chosen because they are highly active for specific parts of the water-splitting reaction: rhodium-chromium oxide for the hydrogen evolution step, and cobalt oxide for the oxygen evolution step.
When co-catalysts are added into a photocatalyst they usually become randomly distributed in the photocatalyst material. Photodeposition makes it possible to instead direct the distribution of the co-catalysts at the proper facets on the surface of the photocatalyst particle. By this method, Domen's team was able to create a photocatalyst with ideal distribution of co-catalysts on different facets; some of rhodium and chromium, and some of cobalt.
a: Time courses of H2 and O2 evolutions on three different photocatalysts;
b: AQY(EQY) at several wavelengths.
The theory is that there exists electric field inside of well-crystalised photocatalyst particles, and photoexcited electrons and holes are carried to different facets through a few-hundreds nanometre length. The electrons arrive at hydrogen evolution co-catalysts consisting of rhodium and chromium immediately produce hydrogen molecules by using proton, and holes arrive at oxygen evolution co-catalysts consisting of cobalt produce oxygen molecules by using hydroxyl anions.
After manufacturing this photocatalyst and repeatedly testing the reaction to check reproducibility, Domen's team had found what they were looking for. It has the highest AQY values recorded for any water-splitting photocatalyst – as high as 95.9% under near-UV light with a wavelength of 360 nm.
A panel with only 1mm of water was capable of rapid hydrogen and oxygen production without the need to stir.
Accessing the visible spectrum
These super-high AQYs are a confirmation of something important: a particulate photocatalyst can drive the overall water splitting reaction at a very high QY. In fact, Domen's photocatalyst can split water almost as efficiently as Nature's very best equivalent: photosynthesis.
Or at least, this is true under certain circumstances. The difference is that in photosynthesis, photons are utilised across a very wide range of frequencies, throughout the spectrum of visible and near-UV light, in the water splitting reaction. In comparison, Domen's version of the SrTiO3:Al photocatalyst works well under near-UV, but its AQY decreases for photons in the visible region: the AQY of the catalyst at 380 nm – the border of UV and visible – is only 33.6%, that is due to the absorption of only UV light.
The photocatalyst under simulated sunlight gives an STH of only 0.65%. This isn't a touch on the 5-10% target, but it was never going to be. The problem is that the STH obtainable from SrTiO3:Al photocatalysts is limited to less than 1.4% even if the AQY reaches 100% at all wavelength regions below 380 nm.
Simulated electric field, and e- and h+ distribution inside two-dimensional model structure.
This is because the photocatalyst being used only absorbs UV light. Domen's catalyst was getting close to an AQY value of 100%, which demonstrates that other photocatalysts with visible light absorption may be able – through tweaking the composition and distribution of co-catalysts – to see similar increases in their AQYs.
Non-oxide photocatalysts
The next steps in this research are to perform the same process for visible-light-active water-splitting photocatalysts. Some non-oxide photocatalysts such as Ta3N5 and Y2Ti2O5S2 have been reported as effective candidates.
Currently, these photocatalysts exhibit AQYs around 0.1%. However, Domen is optimistic that if improvements can be made to these catalysts on a similar scale to SrTiO3:Al then they could easily reach and exceed the 5-10% STH target. His laboratory is, in his own words, "committed to developing such innovative and essential photocatalysts".
Domen's most recent research is not just promising – it is exciting. It stands as a proof concept for water-splitting using photocatalysts using just sunlight, and a signpost towards truly clean hydrogen production.
Personal Response
Can you explain in more detail how it will be possible to apply what you have learned from SrTiO3 to the photocatalysts which are more active under visible light?
To develop photocatalysts with 5-10% STH based on what we have learnt from SrTiO3:Al, it is necessary to use a wide range of visible light responsive photocatalysts such as Y2Ti2O5S2 and Ta3N5 and to devise suitable preparation methods of highly crystalised, low defects density and suitable size of the particles. Then, suitable co-catalysts for water splitting on those particulate photocatalysts must be developed. It is also necessary to develop a water splitting panel and total system of solar hydrogen production, including an efficient hydrogen separation membrane, all which must be scalable, low cost and adhere to safety standards.
Hydrogen? Just add water and sunlight was last modified: July 31st, 2020
'aluminium-doped strontium-titanium oxide'
apparent quantum yield
future fuel
Kazunari Domen
photocatalyst sheets
photodeposition
semiconductor materials
Shinshu University
SrTiO3:Al
ultra-clean fuel
• Takata, T; Jiang, J; Sakata, Y; Nakabayashi, M; Shibata, N; Nandal, V; Seki, K; Hisatomi, T; Domen, K. (2020). Photocatalytic Water Splitting with Quantum Efficiency of Almost Unity. Nature, 581:411–414. doi:10.1038/s41586020-2278-9.
• Goto, Y., Hisatomi, T., Wang, Q., Higashi, T., Ishikiriyama, K., & Maeda, T. et al. (2018). A Particulate Photocatalyst Water-Splitting Panel for Large-Scale Solar Hydrogen Generation. Joule, 2(3), 509-520.
• Hisatomi, T., & Domen, K. (2019). Reaction systems for solar hydrogen production via water splitting with particulate semiconductor photocatalysts. Nature Catalysis, 2(5), 387-399.
• Lyu, H., Hisatomi, T., Goto, Y., Yoshida, M., Higashi, T., & Katayama, M. et al. (2019). An Al-doped SrTiO3 photocatalyst maintaining sunlight-driven overall water splitting activity for over 1000 h of constant illumination. Chemical Science, 10(11), 3196-3201.
• Wang, Q., Hisatomi, T., Jia, Q., Tokudome, H., Zhong, M., & Wang, C. et al. (2016). Scalable water splitting on particulate photocatalyst sheets with a solar-to-hydrogen energy conversion efficiency exceeding 1%. Nature Materials, 15(6), 611-615. doi:10.1038/nmat4589.
• Wang, Q., Hisatomi, T., Suzuki, Y., Pan, Z., Seo, J., & Katayama, M. et al. (2017). Particulate Photocatalyst Sheets Based on Carbon Conductor Layer for Efficient Z-Scheme Pure-Water Splitting at Ambient Pressure. J Am Chem Soc, 139(4), 1675-1683. doi:10.1021/ jacs.6b12164.
• Wang, Q., Nakabayashi, M., Hisatomi, T., Sun, S., Akiyama, S., & Wang, Z. et al. (2019). Oxysulfide photocatalyst for visible-light-driven overall water splitting. Nature Materials, 18(8), 827-832.
• Wang, Q., Okunaka, S., Tokudome, H., Hisatomi, T., Nakabayashi, M., & Shibata, N. et al. (2018). Printable Photocatalyst Sheets Incorporating a Transparent Conductive Mediator for Z-Scheme Water Splitting. Joule, 2(12), 2667-2680.
• Wang, Z., Inoue, Y., Hisatomi, T., Ishikawa, R., Wang, Q., & Takata, T. et al. (2018). Overall water splitting by Ta3N5 nanorod single crystals grown on the edges of KTaO3 particles. Nature Catalysis, 1(10), 756-763.
Behind the Research
Dr Kazunari Domen
Kazunari Domen received a Ph.D. (1982) Honours in Chemistry from the University of Tokyo. He has been Professor since 2004 and University Professor of The University of Tokyo since 2019. He was appointed Special Contract Professor at Shinshu University in 2017.
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
4-17-1 Wakasato, Nagano-shi, Nagano 380-8553, Japan
E: domen@chemsys.t.u-tokyo.ac.jp
E: domen@shinshu-u.ac.jp
T :+81-26-269-5225
W: http://www.domen.t.u-tokyo.ac.jp/english/index_framepage_E.html
Prof Domen's group explores photocatalytic water splitting and develops water splitting panels for renewable hydrogen production on a large scale.
ARPChem Project of New Energy and Industrial Technology Development Organization (NEDO; METI)
Associate Prof T. Hisatomi, Shinshu University
Prof Y. Sakata, Yamaguchi University
Prof. A. Kudo, Tokyo University of Science
DOI: 10.32907/RO-115-158161
Create Harvard Reference
Domen, K. (2020). Hydrogen? Just add water and sunlight Research OUTREACH, 115. Available at: https://researchoutreach.org/articles/hydrogen-just-add-water-and-sunlight/ (Accessed YYYY/MM/DD)
(CC BY-NC-ND 4.0)
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redpajama_set_name: RedPajamaCommonCrawl


I finally satisfied my salted egg yolk crab craving yesterday! It was at this really obscure corner near Outram MRT station opposite of Chinatown and had an odd name "Swis Ling". I first knew about this place back in uni days when my friend Shaun brought us there for lunch one day and we discovered the joys of butter pork ribs. I first wrote about that on Hungrygowhere on Feb 2010, imagine!
Well, Scott mentioned that they also do crabs, and are pretty decent at it too. I was definitely interested when he said they do the Salted Duck Egg Yolk version, with quite rare in Singapore. I had asked a few times around for where I can find them, and the answers had been rather scattered and seems like I had to queue very long for it, so mehh.
But finally, there's one just right near our office and there is no queue on weekdays and no exorbitant prices, selling salted egg crab. Is that even possible?
This plate right here says yes. (And it arrived within 15 mins) The salted egg crab was really nice, and coated with the grainy salted egg butter sauce with bits of roe all around. I usually don't like roe, but those were so fresh and scattered around I did not realize it was roe until I asked about it later (about 5 mouthfuls of roe kind of later). Scott says it is very fresh cos the roe is orange! I agree. The crab meat was also juicy and fresh.
We also ordered a chili crab to counter balance! The classic signature of singaporean crab, the chili sauce was not spicy at all but more like the tomato egg gravy with a mild taste of chili, which makes it really acceptable. Heavenly when fried mantou buns were dipped in them!
Next, I also ordered 2 pieces of Otak because I've tried it before and it was not too bad. Well, yesterday's one was better than that. It was awesome. Look at the large amounts of chunky fish flakes inside, it's like eating mashed fish without bones and just rightly spiced to be very tasty. It came in a weird shape (bigger, wide, short and thick), but who cares as long as it is nice. Heh. Definitely recommend.
And here's the entire set save for the omelette that we ordered later! Guess what, for this whole set, inclusive of 8 honey lemon drinks and 5 rice, the bill only came up to $164.30. That means 20.5 per pax. For 2 crabs, large butter pork ribs, large baby kailan, 2 otah, 10 mantous and a fu rong omelette, it is friggin cheap by singapore's rising living standards.
It is completely alfresco and it feels like malaysia, which is perhaps why the food tastes so good. Other than Ban Leong seafood's chili crab which is better, I definitely know where to go next time for my salted egg crab fix.
Hi Alexis , Mdm Ng ask me to help her update their restaurant number info to 62246110 as their new number because of the 1 year renovation many thought they moved. Pls help , this means a lot to her. thanks a million in advance!


The fundamental question is that can you debar women from worshipping Lord Ayyappa? This is a question. And these women are keeping the pictures of Ayyappa at home and they are worshipping. When a picture or anything similar to that is kept in the house, what does it mean? Ayyappa is present and he is present during their menstruation. What is the difference between one idol in Sabarimalai and so many pictures at home?
I have an email before me which has to be replied. I started replying, came here in between. It is that I should find a solution to the problem of women being permitted or not to Sabarimalai.
Our tradition says that the deity is supposed to be a celibate, austere person or God. And His celibacy will be disturbed when women during their middle age, say from ten to fifty, when they have periodical menstruation happen to visit Him.
The matter has assumed serious proportions. The advocates and the petitioners say their life is in danger and some of the writers and others amongst the women have said "This is an area where court and others should not interfere etc. Why did it become necessary for the court to interfere there? These matters should be discussed and decided upon by sober people who have the wisdom as well as the austerity to deal with any kind of a custom that we have.
Any temple is erected by man. The practices are instituted by man. Man's own mind has invented mantras, tantras, the different forms of worship, the idols, their history, their shapes, the disciplines with which the worship should be done; all have their genesis in the human mind. Is it right to say that our old people had wisdom and austerity to lay down all these and we people are nobody to tamper with them? You don't tamper with them, deal with them in an even much better manner.
Study the whole genesis of worship, God, scriptures etc. Alphabet is evolved by man, the Vedas are all expressed in alphabet. So, were not the Vedas then written or evolved by human beings? Why is it that Vedic authors were human and practitioners of Vedas are not sufficiently human? Can you take such a stand? So this is a point where I have very firm views.
We are worshipping God considering Him to be superior to human beings. The excitable nature of man cannot be grafted to God whom we worship. We worship God to get rid of all our failures and to become pure. We have the story of Ganesha. When Parvathi proposed to organize a marriage for Ganesha, Ganesha said, "My dear mother, I will marry only one who is as good looking as you are. Only when I find one, I will get to marry." Then Parvathy said, "Very good. Go and take your seat in the Ashwattha tree, at the bed of it, where people will come after their bath wearing wet clothes, so you can see them in their natural comeliness. If and when you find somebody acceptable to you like me, I will get you married." Lord Ganesha continues to sit there even now. He has not found somebody suitable to marry. Is Ganesha excited by the sight of young girls, one of whom he is looking for? So Ganesha has this history.
Ayyappa has another history. The question here is that if women or girls upto the age of ten and after the age of fifty can see Lord Ayyappa, then the others also can see and worship. But going to that mountainous temple is a little difficult and arduous and it is said to be associated with a 41 day vow of celibacy. Now, the 41 days to have freedom from menstruation for women is not possible or thinkable. Maybe that is the reason why middle aged women were not going there. If that is the idea and we have opened the temple other than during makara-sankrama etc. can we not have a specific period where women can come, have vision exclusively? Let no men go there, because to ensure the safety of women in such a crowded place is impossible. So, can we not arrange matters in such a manner that women are allowed during a certain period and they alone will come? Whether they will come properly or improperly is not something for men to decide.
We are all born of women. Whatever women are incapable of doing, specially in these matters, I don't think we can trust ourselves, men. Menstruation is a very natural process. We are all born because of that. Why are we looking at it in a wrong manner? I think doctors will be able to explain what is menstruation, and what are the effects on the body and maybe on other factors.
Can we not trust our women? "This is the 'vrata', austere vow that is required for the place. Please make sure that you observe." Are not they going to the other temples? Do they go while they are having menstruation? If they will avoid going to the temple during menstruation and menstrual disturbances, they will not go to Sabarimalai also. Where is the trouble?
The fundamental question is that can you debar women from worshipping Lord Ayyappa? This is a question. And these women are keeping the pictures of Ayyappa at home and they are worshipping. When a picture or anything similar to that is kept in the house, what does it mean? Ayyappa is present and he is present during their menstruation. What is the difference between one idol in Sabarimalai and so many pictures at home? The only point is that, that idol is located in a mountainous shrine and you can have something very special about it. But tell what is special and leave the practice to the women.
Is there anyone in this world who can say "I know more than my mother in the matter of female disciplines and the rest"? This is where corruption in our practices and custom is creeping. Any society is bound to have corruptions in practice. One man has written a letter to me saying this Lord Guruvayoorappan, Guruvayur Temple, people say that it is a Sri Krishna idol. Is it a Sri Krishna idol or a Maha Vishnu idol? I have been telling for many years now whenever I get an opportunity - Guruvayur temple is not a Krishna temple. It is a Maha Vishnu temple. And the idol is said to have been worshipped by Krishna's parents, Devaki and Vasudeva. Now, Devaki and Vasudeva are cleverly forgotten and the legend is also forgotten and they call it a Krishna temple. And this is done by a Devaswom Board, a Devaswom committee which is appointed by a democratic government where they what everything to be termed, to be judged in terms of reason etc.
In our constitution, there is a clause. There should be a scientific thinking, promote scientific thinking and a rational temper in all matters. Now it is under that constitution that our government, MLAs, MPs and the others assume their office, their seats and directly under their nose, this blatant lie is being propagated and nobody has been able to change it.
Do you want our great Sanātana Dharma whose different forms are there in temples and other customs and ways, do you want it to be pure and sustaining or do you want it to be eaten up? Similarly, after Makara-sankranti, the only day in which the Lord Ayyappa of Sabarimalai is anointed with his full princely dress, ornaments, swords and other things. People go there only to see him decked with the traditional ornaments which are specially brought for that day. That day, suddenly people were clandestinely hoisting a flame calling it 'makara-jyothi'. All the people started looking at false pseudo 'makara-jyoti' showing their back to Ayyappa. They are showing their back and Ayyappa is forgotten and 'makara-jyoti' has taken precedence.
And what is this 'makara-jyoti'? The Kerala High Court finally asked "Tell us whether it is a human flame or a divine flame." The Devaswom Board started speaking this and that and they tried to escape. They forced the Devaswom to give an answer. And they said it is hoisted by the Devaswom Board. So they were keeping the whole devotees under a delusion. And then because of the mass requirement, High Court said, "Okay. You hoist it by a priest of the temple and debit the expenditure to the Devaswom. Now does it require a Court to say this? Are we such liars and such incapable people that our religion should be interfered with for its need of propriety by the Court?
If you say that the court is a secular court, why should they deal with a religious matter? But the concept of the court is whenever there is a dispute or an indecision about any matter, suppose somebody says Arabian sea has been set fire to, they will have to say, they will have to come up with an adjudication. So you cannot blame the court. Whenever a matter is taken before the court, they have to adjudicate because the citizens can go only to the court. Sometimes government, parliament, assembly, all of them err in their judgment. The only way it can be redressed is by going to the court. Just see?
They say an idol is installed, Padmanabha Swami is installed by people and if necessary, you can take out the idol and conduct a rejuvenation. But the offerings received in the name of Padmanabha Swami are kept in a particular room. That room should not be opened because opening that room will undermine the sanctity and divinity of the idol. How can we permit this kind of an argument? But mostly it goes under tradition and custom. There are very rare people who have the austerity, confidence and clarity to say it is not so. Our Hindus should themselves be able to resolve this problem. Instead of that, because of mass devotional movement behind this, it finally goes to the court. It is a very, very deplorable situation.
Our shastras are very clear. We have enlightened people also and ultimately, the Hindu thinkers are never given an opportunity to solve the Hindu problems, dhārmik problems. They are taken to the court and you say court should not interfere. What else will they do?
So, Kerala High Court asked the Travancore Devaswom Board "Tell us whether it is a humanly raised flame or a divine flame." Such a question had to be raised by a judge, can you imagine? Shamelessly the Devaswom board was trying to wiggle out of it. So I was just mentioning the subject and I am writing a reply. I thought of sharing it with you.


text:
Joy, for now
Opera Gallery Singapore
To coincide with Singapore Art Week, Opera Gallery is pleased to present Joy, for now, a selection of new works by British artist George Morton-Clark, in his first solo exhibition in Singapore.
Born in 1982 in Tooting, South London, British artist George Morton-Clark utilises oil, acrylic, and charcoal on unprimed, usually large canvases to bring his characters to life. With a combination of bold images and strong colours, his paintings and sculptures are immersed in an abstract atmosphere of energy and liveliness. Drawing his inspiration from films, music, his travels and pop imagery, he combines abstract elements with figurative, working in expressionistic techniques. He is wary of complacency and constantly strives to move his work in a new direction, resulting in his art becoming a mixture of a variety of styles, each time challenging the way that we experience it. Morton-Clark's artworks draw on the nostalgic and universal connection that we all have to his cartoonish figures in a quest to connect to everyday life. While the characters evoke a collection of emotions and expressions from surprise to fear, we are engulfed in a state of curiosity, which the artist responds to with an ambiguous simplicity. His artwork is simply a gift to the viewer, a small window of relaxation and simple enjoyment that can use to their heart's content.
Join us in an exploration of George Morton-Clark's world of infinite possibilities, and together let's rethink the simple, everyday joy, for now.
GEORGE MORTON-CLARK, Bear Temper, 2021
Charcoal, oil, pastel, acrylic and spray on canvas
150 x 120 cm | 59 x 47.2 in
GEORGE MORTON-CLARK, Badly Drawn Superhero, 2021
Oil, acrylic, charcoal, pastel and spray on canvas
170 x 150 cm | 66.9 x 59 in
GEORGE MORTON-CLARK, Counterfeit Ready, 2021
GEORGE MORTON-CLARK, End of Red, 2021
Oil, pastel and spray on canvas
GEORGE MORTON-CLARK, Full Moon Dichotomy, 2021
100 x 85 cm | 39.3 x 33.5 in


What can you do to make sure you will enjoy your cigars to their fullest potential? "if you are going to truly enjoy smoking a cigar, it is worth your time to light it slowly, and light it properly." Turns out the same wisdom goes when actually smoking it, too.
Enjoying cigars is relaxing way to spend some solid "me" time, in addition to being my line of work. But pacing myself has paid unexpected dividends when it comes to truly enjoying them: just as I do not gulp scotch, I don't rush my cigar; so I recommend you take your time and savour it. I bet you will find that many cigars you thought were not as good and do not live up to the hype, actually take on a whole new flavour-rich life of their own.
There are two ways that tearing through a premium cigar is doing you and your palate more harm than good…first, it is about smoking time. Maybe you find that you can blast your way through a robusto in under 30 minutes, or worse, many of your most-recently smoked cigars have had a bitter taste. Either could be a sign that you are puffing too hard, too fast. If that is happening to you, slow down. When considering how often to puff a cigar, a good rule of thumb is to be patient and hit on the cigar roughly once a minute or so. It lets the cigar burn a little cooler, and allows for your senses to pick up the flavours and aromas the cigar maker blended. Bonus: taking fewer puffs can also help make full-bodied cigars that much more manageable.
Smoking a cigar too fast can prematurely char the filler tobacco, and cause an ashy or bitter taste. And when you make it down to the nub in a more reasonable amount of time, the heat will not ruin your smoke – or your taste buds.
In addition to being mindful about how often to puff a cigar, try rotating your cigar as you smoke it too; smoking with the same side always up can cause an uneven burn. By rotating your smoke, the heat is more evenly distributed throughout the surface area of the cigar – creating a better burn, and a noticeable improvement in flavour.
Even the pros take a break between cigars. When testing new blends, it is not uncommon or unreasonable for cigar makers to smoke cigars by the dozen, and more than one at a time. But even the blenders know the senses need a time-out – and will take a few minutes away between cigars to cleanse the palate with a drink, so their taste buds do not go down in flames.
You wanted an unexpected benefit to smoking more slowly? Listen to your wallet. If you smoke 5 cigars in an afternoon at $25 a piece, that is a lot of premium cigars up in smoke. Slow it down – pace yourself, and try getting 90 minutes out of that Churchill. Now you are down to 3 cigars in an afternoon – and you might actually taste them all. You will still get the most out of each smoke, but now you are buying a box of cigar a little less frequently, saving some coin and consistently enjoying your daily cigars.
Choose the right sized cigar to get the job done. Short on time? Go for a smaller RG or shorter cigar rather than powering your way through a 6×60. Got an evening with time to kill? Skip that tin of cigarillos and go big, but smoke slow – no one wins in a race to the nub. Pacing yourself while smoking a cigar makes a difference – a huge difference. You can have yourself a rich, robust and flavourful cigar experience, or a "meh" experience…and I think you certainly deserve the former. Instead of laying down a smokescreen the next time you light up, try to enjoy all of the flavours that cigar is meant to have. Relax – slow down. There will be more cigars to smoke and enjoy.