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The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.
The term precision medicine describes the idea of providing effective treatment based on a patient's molecular make up. Recent advances in molecular diagnostic tools and handling of large data sets allow for the stratification of patients based on e.g. genetic or protein information and make it possible to provide tailored treatment for these sub-groups1. Here we describe how epitope information, exemplified by binding of the therapeutic antibody eculizumab on its target complement component 5 (C5), can be used to guide treatment.
The complement system is an important part of the human innate immunity. It fulfils an important role in fighting bacterial infections and homeostasis and provides a link between innate and adaptive immune response2. Such powerful functions require a tight regulation to prevent the complement system from attacking the host's cells. Paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS) are two disorders associated with a malfunctioning complement system. PNH is characterized by blood cells deficient of glycosylphosphatidylinositol (GPI)-anchored proteins, which protect cells from complement attack, caused by an acquired mutation in hematopoietic stem cells3,4,5. This consequently results in intravascular haemolysis, which in turn leads to anaemia and thrombosis4,5,6. Atypical HUS is a renal disease that is characterized by an unregulated activation of the alternative pathway of the complement resulting in thrombosis in kidney capillaries and renal failure7,8.
The monoclonal antibody eculizumab (Soliris®) is the first FDA-approved therapeutic antibody for treatment of PNH2,4,5,9 and aHUS7,10. Eculizumab is a humanized monoclonal antibody targeting C5. The suggested mode of action is that eculizumab binding to C5 prevents the entry and subsequent cleavage of C5 by C5 convertase7,11. C5 is a central protein in the complement system cascade that is common to all three pathways. This makes it attractive as a therapeutic target. By inhibiting C5 cleavage the common downstream cascade can be disrupted independent of the activating pathway, i.e. no C5b-9 formation (membrane attack complex, MAC) and generation of the anaphylatoxin C5a occurs2,5. At the same time, the upstream pathways resulting in generation of C3b are kept intact and thus pathogen clearance via C3b-mediated opsonisation is not impaired4,5. Besides the approved treatment of PNH and aHUS, eculizumab has also proven effective in some cases for treatment of other conditions, like myasthenia gravis12,13, neuromyelitis optica14,15, membranoproliferative glomerulonephritis16,17, catastrophic antiphospholipid syndrome18,19,20 and prevention of rejection after renal transplants21,22,23.
Despite the proven relevance of the drug, reports on non- or poorly responding patients have been published24,25,26,27,28. Among them a study on Japanese PNH patients which carried a mutation in the C5 gene, which changes the C5 protein so that it can no longer be bound by eculizumab24. This highlights the need for an exact determination of eculizumab's epitope on C5. Such knowledge would enable the development of genotyping kits for the prediction of responsiveness of a patient to eculizumab treatment.
Different efforts have been undertaken to identify the binding site of eculizumab on C511,29,30. In an earlier report to the Japanese Pharmaceutical and Food Safety Bureau29, eculizumab was reported to have a conformational epitope consisting of three distant amino acid stretches covering residues 822 to 826 (DVFLE), 879 to 883 (KSSKC) and 930 to 933 (VPEG). Later, Zuber et al.7 only refer to KSSKC as eculizumab's epitope. In a recent publication30 based on transmission electron microscopy (TEM), eculizumab was found to interact with the MG7 domain with R885 and the KSSKC peptide being part of the binding site. While the present study was under review the crystal structure of C5 in complex with the eculizumab Fab fragment was solved11. While accurately determining the region of binding, the resolution of the co-crystal structure did not allow an unambiguous determination of intermolecular interactions at residue level.
Here, we used an epitope mapping strategy based on deep mutational scanning of surface-displayed protein domains to explore in detail the structural epitope properties of eculizumab. While high-resolution X-ray crystallography of the antibody complex still represents the gold standard for epitope determination31 other epitope mapping methods have been developed, like peptide scanning32, alanine scanning33 and several combinatorial library techniques34 that are applicable to a wider range of proteins and offer higher throughput. The approach presented here is based on the gram-positive bacterium Staphylococcus carnosus (S. carnosus)35,36, which proved to be advantageous for display applications due to its high mechanical resistance allowing for cell sorting applications and the lack of extracellular proteolytic activity37,38. For the first time, staphylococcal display was used for mapping of a structural epitope by displaying randomly mutated protein domains on the surface of S. carnosus. The combination of random mutagenesis and surface display of protein domains allows a more thorough analysis of the mutational space under native-like conditions in contrast to other high-throughput mapping techniques like peptide arrays and alanine scanning.
The presented workflow (Fig. 1a) includes the identification of a binding domain, creation of a mutant display library, identification and sorting of surface expressed non-binding domain mutants using flow cytometry and sequencing of them. To confirm the identified positions the mutants were produced as full-length proteins in mammalian cells and functionally analysed for haemolytic activity and binding to the antibody. The identified C5 residues involved in the binding to eculizumab were matched against reported germline variations of relevance for the prediction of responders to treatment with this therapeutic antibody, which constitutes the stratification part of the workflow (Fig. 1b). Comparing the patient's gene sequence with the epitope of available drugs enables stratification into treatment groups. Our work outlines a general strategy for stratification of responders and non-responders for treatment with therapeutic antibodies and more specifically analyses the underlying molecular mechanism for patients' response to the therapeutic antibody eculizumab.
Eculizumab binds to MG7 domain displayed on Staphylococci
The complement factor C5 has been structurally characterized and found to consist of twelve domains39,40. Each of the twelve domains (colour coded in the Fig. 1a) was cloned and expressed using a Gram-positive bacterial surface expression system (Supplementary Fig. S1). This allowed binding analysis of the monoclonal antibody eculizumab using a fluorescently labelled secondary antibody and flow sorting of the resulting bacterial cells with the respective domains exposed on the surface (shown schematically in Fig. 1a). The binding analysis showed that the monoclonal antibody binds exclusively to the domain MG7 (residues 822–931) expressed on Staphylococci, while all other eleven domains were shown not to interact with the antibody (Fig. 1c).
Loss of binding mutations identified from random mutagenesis library
Having identified the MG7 domain as the binding domain for eculizumab, a random mutagenesis library of this domain was constructed by error-prone PCR (as shown schematically in Fig. 1a. The size of the staphylococcal library was 1.42 × 104 clones and sequencing of 96 randomly picked clones revealed a mutation frequency of 1.4 nucleotide mutations per gene (data not shown). The albumin-binding protein (ABP), expressed in fusion with the respective mutated domain before the membrane anchoring domain, was used with labelled human serum albumin (HSA) to detect surface expression of the domain. Mutations within MG7 leading to overall misfolding of the domain or introduction of stop codons were expected not to be secreted nor properly anchored to the cell surface34, resulting in a decrease or absence of ABP signal for these, making it possible to avoid these during flow sorting. Clones that showed a retained high HSA signal, but a reduction in eculizumab binding were sorted in two successive rounds of flow cytometric cell sorting as indicated in Fig. 1c. After cultivating this population of enriched mutants under selective conditions on an agar plate, colonies from the sorted pool were picked and subjected to sequence analysis and characterization. Out of 90 individual clones, 71 coded for unique protein sequences with the majority containing several amino acid mutations. To facilitate data analysis and in order to be able to draw unambiguous conclusions about the contribution of a residue to antibody binding, clones containing multiple amino acid mutations were excluded from the analysis. 15 clones were identified encoding for a single amino acid mutation (Supplementary Fig. S2). Mutations involving the amino acids cysteine, glycine and proline were omitted from the analysis due to the fact that such amino acid changes often result in disruption of the overall conformation34. One of the clones discarded due to the mutation coding for a cysteine (R885C) had earlier been reported as a mutation present in patients24 and was therefore included in the subsequent analysis. This resulted in the identification of 11 clones (I829K, Q854L, F855I, R885C, R885H, K887N, K887R, K887T, V896E, W917L, F918S) with a single mutation in the MG7 domain.
The 11 identified clones were analysed individually by flow cytometry for their binding to eculizumab. The analysis confirmed that they all had reduced binding to the monoclonal antibody compared to the wild type control (Fig. 2a). Some mutations, such as F918S and K887T, reduce the capability of binding to eculizumab, while others, such as Q854L, R885C/H and K887N, seem to eliminate binding completely. Interestingly, the three different mutations found at position 887 all showed reduced binding, but the degree of binding varied considerably depending on which amino acid it was changed to (asparagine, arginine or threonine). In summary, the results from the eleven clones suggest that eight different amino acids in the C5 domain are involved in the binding to the therapeutic antibody eculizumab.
Mammalian produced full-length C5 mutants show decrease in binding to eculizumab
The wild type C5 protein and nine mutants identified above including R885C and one of the K887 mutants (K887N) were expressed and purified from transiently transfected CHO cells. A C-terminal protein C peptide tag41 enabled efficient affinity purification using the HPC4 monoclonal antibody. After purification, the purified C5 proteins were analysed by SDS-PAGE (Supplementary Fig. S3). The protein gel displayed two clear bands of the alpha (104 kDa) and beta chain (73 kDa) confirming a proper maturation by specific protease cleavage of pro-C5. The ability to purify both chains using the C-terminal tag of the beta-chain indicates that the two fragments are covalently interacting through native cysteine bridge.
The nine mutant C5 proteins together with the wild type C5 were analysed for binding to eculizumab by sandwich ELISA. The analysis confirmed that many of the mutants, except I829K and V896E, showed reduced binding (Fig. 2b). Although the effect for F918S is relatively low and the decrease in binding for W917L is non-significant, we decided to analyse also these mutants further. Most dramatic effect in binding to eculizumab could be attributed to the R885 mutants with almost complete loss of reactivity in the ELISA assay.
Eculizumab cannot inhibit the haemolytic activity of the C5 mutants
The haemolytic activity, i.e. the ability to elicit C5b-9 formation, was analysed for the recombinant C5 variants that showed reduced binding to eculizumab in ELISA to exclude that the drop in binding is due to a major structural change brought about by the mutation. The analysis shows that all mutants are able to induce C5b-9 formation in a concentration-dependent manner (Fig. 3a), although some, like R885C, do this to a lower degree than wild type C5. The lower activity of W917L could be attributed to the fact that this residue is likely to be involved in convertase binding42. When adding 3.7 nM eculizumab to this activity test, C5b-9 formation was inhibited in wild type C5, while mutants Q854L, R885C, R885H and W917L showed a nearly unchanged activity and F855I, K887N and F918S displayed a clearly lowered eculizumab effect (Fig. 3b and Supplementary Fig. S4). The decreased effect of eculizumab on F855I is more distinct in Supplementary Fig. S4 showing that a higher amount of eculizumab is needed to inhibit F855I compared to WT C5. When 3 nM Ornithodorus moubata complement inhibitor (OmCI), a tick protein43,44 that does not compete with eculizumab for binding to C530, was added instead of eculizumab, C5b-9 formation was inhibited to the same degree as WT for all C5 variants (Fig. 3b,c for R885C). All analysed mutants showed a significantly reduced haemolysis when incubated with OmCI compared to eculizumab (Fig. 3b).
The six identified residues co-locate with the epitope regions identified by crystallography
The combined results from the binding and haemolysis experiments suggest that six residues are involved in binding to eculizumab, i.e. Q854, F855, R885, K887, W917 and F918. These residues are widely spread over the MG7 protein sequence (Fig. 4a) and therefore a structural analysis was done using the published co-crystal structure of C5 and an eculizumab Fab (PDB5I5K)11. The analysis showed that all six residues are brought together into a single site on the β-sheet in the folded C5 MG7 domain (Fig. 4b). All six residues fall into the three contact regions (residues 851–858, 882–888 and 915–920) identified by Schatz-Jakobsen et al.11 based on a less than 5 Å-distance to the Fab fragment. Further analysis revealed that, except for F855I, all five residues are more than 25% surface exposed (Fig. 4c) rendering them accessible for the antibody (Fig. 4d).
Naturally occurring germ line mutations of C5 located in epitope
To identify possible germ line mutations in the C5 gene, three different databases (NCBI dbSNP, 1000genomes and ExAC) were searched for missense mutations in MG7. Altogether 32 naturally occurring variations in the MG7 domain were found, listed in Table 1 together with the positions of the six identified epitope residues. Two of the mutations found to abolish binding of eculizumab to C5 (R885C and R885H) were found in the database query originating from a report24 on poor response to eculizumab in PNH patients. In addition several genetic variations were identified in close proximity to the suggested epitope and within the 5 Å region described above11, which could affect the binding of the drug to a lower degree.
W917 is specific for human C5 and is critical for eculizumab binding
It has been reported that eculizumab is specific for human C5 and that the lack of reactivity of orthologous C5 proteins has prohibited use of such animal models5. In Supplementary Fig. S5 the protein sequence for the epitope residues are aligned in nine different species, including primates, rat and mouse to try to explain this lack of binding. While F855 is completely conserved in all nine species, only the chemical properties of the residues at positions 887 and 918 are conserved. Residues Q854 and R885 are conserved between humans and primates, while W917 is unique for humans. All other analysed animals show a serine instead of tryptophan at position 917. Interestingly, analysis of the binding properties and haemolysis inhibition of a W917S mutant (Supplementary Fig. S6) demonstrates that this mutant has significantly decreased binding activity, thus explaining why eculizumab is non-functional in these animal models.
This work presents six residues on the complement protein C5 that are essential for the binding and function of the therapeutic antibody eculizumab and suggest that other means of treatment should be considered for individuals carrying mutations in these positions. All 12 domains of C5 were successfully expressed on the cell surface of S. carnosus and domain MG7 was identified as the binding domain of the therapeutic antibody eculizumab. A deep mutational scanning of MG7 was performed to determine the amino acids involved in eculizumab binding. After excluding multiple mutations and mutations to or from cysteine, glycine and proline that are likely to disrupt the protein structure34, eight residues were identified, which after functional analysis were condensed to six essential residues required for binding and function of the antibody. Although these residues are localized on different strands of the MG7 domain, they cluster to a single, contiguous site in the folded protein, suggesting that the epitope of eculizumab on C5 is dependent on tertiary structure.
This hypothesis was confirmed by the C5-eculizumab Fab co-crystal structure11 published after submission of this article. Schatz-Jakobsen et al. report three contact regions on C5 within the β-sheet of MG7 comprising residues 851–858, 882–888 and 915–920. Although the resolution of the crystallographic data did not allow the clear identification of single epitope residues, the authors inferred from the existence of binding pockets on the antibody that residues W917, F918 and R885 are involved in binding. All three residues could be confirmed to be essential for antibody binding by the surface display based epitope mapping approach presented here. In addition, three other residues, Q854, F885 and K887, which locate in the first and second contact region, described by Schatz-Jakobsen et al.11, respectively, were here shown to be important for eculizumab binding. Therefore, we regard the approach presented here using independently folded protein domains as a screening platform well suited for detection of contact residues in a structural context.
Our binding assays with full-length C5 variants from mammalian sources have shown that the findings about the eculizumab epitope on domain level on the staphylococcal surface are transferable and relevant for full-length human C5. Analysis of surface exposure reveals that all residues but F855 exceed 25% surface exposure. This might account for the lower degree of haemolytic activity, i.e. a higher degree of inhibition by eculizumab, observed for this mutant in the haemolysis inhibition assay (Fig. 3b). As Schatz-Jakobsen et al.11 we identified residue W917 as an important contact residue in this work. Our mutation studies confirmed the absence of eculizumab binding to a W917S C5 mutant. Sequence comparison showed that tryptophan at this position is specific for human C5. Considering that chimpanzee C5 only differs in two amino acids within the MG7 domain from human C5, W917 being one of them, these findings explain the species specificity observed for eculizumab, which is not able to bind to C5 of any other species but human5. Also R885's involvement in eculizumab binding could be confirmed in this work. The crystal structure suggests that the mutated residues (His and Cys) are too small to fit the binding pocket11. Schatz-Jakobsen et al. also hypothesised that a cysteine mutation at that position could affect the disulphide bridge between residues 856 and 883. We observed a decreased haemolytic activity for the R885C mutant which could be explained by a structural change in the MG7 domain that perturbs interaction of this domain with the convertase42.
The clinical relevance of R885-mutations is highlighted by a study on Japanese PNH patients24 exhibiting poor response to eculizumab. Considering these insights from R885 mutation on eculizumab response, the same is thought to be true for any of the other residues found in this work to be involved in eculizumab binding. Although no germline mutations are reported for these five residues today, mutations have been reported for residues in close proximity to eculizumab as determined by the crystal structure11. Also mutations at these positions are likely to impair the responsiveness to eculizumab. But an analysis of single mutants would be needed to verify this. As shown in the haemolysis inhibition analysis patients with a mutation at F855, K887 and F918 would need a higher dose of eculizumab to elicit the same response as with non-mutant C5, while patients carrying a mutation at Q854, R885 and W917 likely respond so poorly to eculizumab treatment that an alternative treatment should be considered. Consequently, a clearly defined epitope as presented here can prove helpful in the clinics to screen for patients' susceptibility to eculizumab treatment and allows for a more effective treatment.
Moreover, it could be shown that the C5 complement inhibitor OmCI, which binds at a site distinct from eculizumab30, was, in contrast to eculizumab, able to completely prevent the haemolysis induced by the mutant C5 molecules. Considering that OmCI, also known as coversin, was earlier reported as a possible therapeutic30,45,46,47,48 with low immunogenicity45 and is currently undergoing clinical trial and given the results presented here, OmCI could represent an alternative future treatment strategy for patients responding poorly to eculizumab due to a missense mutation in eculizumab's epitope on C5. Taken together our work outlines a general strategy for precision medicine based on detailed epitope mapping and genetic profiling, which should be transferable to several other diseases and therapies and which would allow for correct treatment on those who will benefit while sparing expense and side effects for non-responders.
Materials and Methods
Construction of the epitope mapping library and expression in S. carnosus
The mRNA sequence for C5 was obtained from Source BioScience (Cambridge, UK). The C5 domains according to Fredslund et al.40 were amplified by PCR and cloned into the staphylococcal display vector pSCEM238 (Supplementary Fig. 1) Transformation and library construction in S. carnosus is achieved as described before49. Briefly, the display vector pSCEM2 was amplified in E. coli and purified prior to transformation of S. carnosus TM 300 cells (a kind gift from Prof. F. Götz, University of Tübingen, Tübingen, Germany). S. carnosus cells were transformed by electroporation using a Bio-Rad MicroPulser electroporator at 2.3 kV for 1.1 ms. Transformed cells were grown at 37 °C overnight for protein surface expression. Having identified the C5 MG7 domain as binding domain of eculizumab (produced from CHO cells at Swedish Orphan Biovitrum AB (Sobi), Stockholm, Sweden, M.M.B, Chao Su, Lotta Berghard, Göran Selén, Kerstin Larsson, Joakim Nilsson, Catharina Sterky, Carina Ekholm, Per-Olof Edlund, Charlotte Söderberg Nyhem, P.S., manuscript in preparation), the domain was amplified using a Genemorph random mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) to yield an average mutation frequency of 1.4 mutations per sequence. The vector was purified by phenol chloroform extraction and cloned into S. carnosus as described above.
Antibody labelling and analysis by flow cytometry
Of an S. carnosus overnight culture 2 μL were taken for analysis. The cells were washed twice with phosphate buffered saline containing 0.1% pluronic (BASF, Ludwigshafen, Germany) (PBS-P) before incubation with eculizumab (8.8 nM) for 45 min at room temperature. The cells were then washed with PBS-P and incubated with Alexa 488-labeled goat-anti-human secondary antibody (2 μg/mL) and Alexa 647-labeled HSA (1:1000 dilution) for expression control on ice in the dark for 30 min. After a final washing step the cells expressing the protein domains were analysed in a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). The same protocol was also applied when analysing the identified library clones individually.
Sorting of the random mutagenesis library
The library clones were labelled in the same way as described above. The cells were analysed and sorted in a MoFlo Astrios cell sorter (Beckman Coulter, Brea, CA, USA). The library was sorted for binding escape mutants that show a pertained high surface expression signal but a decreased antibody-binding signal. Two rounds of sorting for binding escape mutants were performed with the sorting gates indicated in Fig. 1c to reduce the risk for false positive clones. The sorted cells were incubated on agar plates and 128 clones picked for sequence analysis.
Production of soluble C5 variants in mammalian cells
The gene for human C5 was cloned into vector pQMCF1 coding for full-length C5 protein C-terminally fused to a tobacco etch virus-protease site and a Human protein C epitope tag (HPC4). C5 mutants were derived from this plasmid by using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). Successful mutagenesis was confirmed by Sanger sequencing. Recombinant wildtype and mutant C5 was expressed in Chinese hamster ovary (CHO) EBNALT 85 cells (Icosagen, Tartu, Estonia) following the manufacturer's guidelines for extended transient expression. After 15 days of cultivation C5 proteins were purified from the cell culture supernatant by affinity chromatography on an anti-protein C affinity matrix (Roche, Basel, Switzerland). Due to low protein concentration, some samples were concentrated using a centrifugal filter (Merck Millipore, Darmstadt, Germany). Protein concentration was determined with ImageJ (see supplementary methods) from a Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. S3). All non-concentrated protein samples had a purity of over 95% as assessed by Coomassie staining after SDS-PAGE (Supplementary Fig. S3).
Binding analysis of full-length, soluble C5 variants
Binding of recombinant wildtype and mutant C5 was assessed by ELISA. A 96-well half area plate was coated overnight with 33.3 nM OmCI (produced at Sobi as described in Bloom et al.50). The next day, the plate was blocked with PBS containing 1% bovine serum albumin (BSA). C5 variants were added at a concentration of 63 nM in blocking buffer. After washing the plate, eculizumab was added at a concentration of 16 nM. Antibody binding was detected with help of an anti-human IgG antibody coupled to horseradish peroxidase (HRP) and Tetramethylbenzidine (TMB) substrate. The reaction was terminated by addition of 2 M sulfuric acid. Absorbance was measured at 450 nm.
Activity analysis of full-length, soluble C5 variants
The ability of WT and mutated C5 to form C5b-9 was monitored by haemolytic activity using antibody (Rabbit anti-sheep RBC stroma, Sigma #S1389) coated sheep red blood cells. 5 × 106 cells in 50 μL GVBp (0.15 mM CaCl2, 0.5 mM MgCl2, 3 mM NaN3, 138 mM NaCl, 0.1% gelatine, 1.8 mM sodium barbital and 3.1 mM barbituric acid, pH 7.3–7.4) were used and the amount of hemolysis was monitored as absorbance at 415 nm. Diluted C5 deficient human serum (1:50) was spiked with C5 (WT or mutant) at different concentrations ranging from 51.2 nM to 6.25 pM. Subsequently, the capacity to inhibit haemolytic activity by eculizumab and OmCI at 100–0.001 nM was monitored using C5 concentrations optimized to normalize haemolytic activity between C5 variants; 0.023 nM (WT), 0.013 nM (Q854L), 0.011 nM (F855I), 8.9 nM (R885C), 0.089 nM (R885H), 0.046 nM (K887N), 0.72 nM (W917L), 1.6 nM (W917S) and 0.089 nM (F918S).
Data analysis
The six identified amino acid mutations were visualized on an existing 3D structure of C5 (PDB5I5K)11 using ICM software (Molsoft LLC, San Diego, USA). For comparison of the six identified residues with naturally occurring germ line mutations, three databases, dbSNP51, 1000genomes52 and the Exome Aggregation Consortium (ExAC) browser53, were searched for missense mutations occurring in the MG7 coding region (residues 822–931). Furthermore, the protein sequence of human C5 was aligned to C5 sequences of chimpanzee (Pan troglodytes), crab-eating macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), cat (Felis catus), wild boar (Sus scrofa), cow (Bos taurus), rat (Rattus norvegicus) and mouse (Mus musculus). The amino acids at the six identified positions were analysed for species conservation.
Statistical analysis
The mean of duplicates or triplicates is depicted in all column charts. Error bars indicate standard deviation. Statistical significance was calculated using GraphPad Prism Software. One-way Analysis of variance (ANOVA) with Dunnett's multiple comparison test and two-way ANOVA with Tukey multiple comparison test was applied for ELISA results and haemolytic activity testing, respectively.
Additional Information
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Geysen, H. M., Meloen, R. H. & Barteling, S. J. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998–4002 (1984).
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S. Laursen, N., Magnani, F., H. Gottfredsen, R., V. Petersen, S. & R. Andersen, G. Structure, Function and Control of Complement C5 and its Proteolytic Fragments. Curr. Mol. Med. 12, 1083–1097 (2012).
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This work was supported by the ProNova Centre for Protein Technology, the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark, Wallenberg Center for Protein Research and the Human Protein Atlas. The Erling-Persson Family Foundation is acknowledged for financial support of the KTH Center for Applied Proteomics.
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Volk, A., Hu, F., Berglund, M. et al. Stratification of responders towards eculizumab using a structural epitope mapping strategy. Sci Rep 6, 31365 (2016) doi:10.1038/srep31365
Further reading
Current and Future Approaches for Monitoring Responses to Anti-complement Therapeutics
Frontiers in Immunology (2019)
Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)
Molecular & Cellular Proteomics (2019)
The rational design of affinity-attenuated OmCI for the purification of complement C5
Journal of Biological Chemistry (2018)
Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
New Biotechnology (2018)
Staphylococcus carnosus: from starter culture to protein engineering platform
Applied Microbiology and Biotechnology (2017)
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Picture this: It's Christmas day, and freshly fallen snow blankets your yard. You cover your eyes while your toddler leads you into the living room, where a $2,245 Peloton stationary bike gifted by your flannel-wearing husband sits unwrapped by your meticulously decorated Christmas tree. A noise that passes off as excitement exits your mouth, and "She's So High" by Tal Bachman starts playing in the background for some reason.
Too much for your imagination to handle? No worries, you can just watch the actual Peloton commercial that made this holiday-themed Black Mirror–esque episode a reality.
As the rest of the 30-second commercial progresses, the woman goes to painstaking measures to document her Peloton journey. "First ride. I'm a little nervous but excited," she announces to her phone's front-facing camera, because, of course, who wouldn't be nervous to start furiously pedaling alone in their living room when the window is wide open for all the neighbors to see? "Let's do this."
Twitter has a way of digging up the best well-intentioned yet horribly executed adverts, only to expose them to the merciless online masses. Harsh feedback to the TV spot (aptly titled "The Gift That Gives Back" on the company's YouTube channel) drove down Peloton's shares by as much as 10 percent, according to Bloomberg. "Our holiday spot was created to celebrate that fitness and wellness journey," Peloton told CNBC. "While we're disappointed in how some have misinterpreted this commercial, we are encouraged by—and grateful for—the outpouring of support we've received from those who understand what we were trying to communicate."
There's so much to pick at: The inherent passive aggression in gifting thousands of dollars' worth of exercise gear to your spouse for the holidays; the way the actress's wide doe eyes crinkle in quiet desperation as she records her daily Peloton routine, and how her voice somehow always has a slight gasp to it—like she constantly cannot believe that she's indeed sitting on a stationary bike; the misogynistic undertones in suggesting that a woman would react in any way other than bewilderment at receiving a literal stationary bike as a present. Still, my personal favorite is the music, because what else evokes those warm fuzzy feelings of a Christmas miracle more perfectly than that one late-'90s rock song that seems to be in every coming-of-age movie?
By the ad's end, the wife is on the verge of tears, but not for the reason that you'd think! "A year ago, I didn't realize how much this would change me," she says to the camera at the end of her homemade Peloton documentary. "Thank you."
Really, the ending is just a more elaborate riff off Instagram's favorite Christmas caption: "It's an avocado … thaaaanks."

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Overview[ edit ] In his own words, Bernays describes engineering consent as "use of an engineering approach—that is, action based only on thorough knowledge of the situation and on the application of scientific principles and tried practices to the task of getting people to support ideas and programs. Their dispassionate approach and methods may be likened to those of the engineering professions which stem from the physical sciences. Consent that is thus engineered is difficult to distinguish in any fundamental way from the consent that supports modern totalitarian governments. Were the manipulated voter to become the normal voter, the government he supports could hardly be said to rest on his consent in any traditional sense of that word.
The Press, as identified with Newspapers, wields immense power in a democratic society. Dickens has called the Press "the mighty engine".
So great is its influence that some have called it the Fourth Estate. Napoleon used to say— "Your hostile newspapers are more to be feared than a thousand bayonets. The most powerful autocrat is forced to take note of public opinion as reflected in a free press.
That is why a regimented press is the instrument of autocracy, and a dictator deprives the press of its freedom.
A free press is the symbol of a free people. An independent, well-informed press is a powerful check on arbitrary governments and irresponsible administrators. For newspapers are agents of the public, which bring to the notice of the people acts of injustice or oppression, or mal-administration that would otherwise have remained hidden away from public knowledge.
They exercise a constant vigilance on the rulers, which is salutary for all. Nowadays it is difficult for the press to be free. Either a newspaper is controlled by some financial magnates entirely and it has to voice their views or it is the mouthpiece of a party, write an essay on freedom of press it must think as the party might direct; or it is under the thumb of the government and in that case its usefulness is reduced and independence compromised.
For whoever controls it, necessarily limits its freedom by his own interests, i. In America, the great newspapers are in the bands of powerful financial syndicates; in England, they are in the hands of capitalists; in Russia they are mostly controlled by the government.
But freedom of the press in each case depends on the way it reflects the will, the purpose of the people as a whole, in preference to that of any class or community or individual. The illusory or unrealistic character of the freedom of the press was seen during Great War.
It is in such times of crisis that the voice of the people should be most clearly heard and not muffled.
Yet it is then that newspapers are completely gagged. Most of these abuses might be checked to the advantage of all concerned, if the newspapers have freedom to criticise authorities and ventilate grievances. Indeed, no government can stifle the voice of the people but at its own peril. The problem, therefore, is—how to ensure freedom of the newspaper.
Legislation, of course, should guarantee the press against interference by the government; this goes without saying- But something more is necessary.
The press under no circumstances should be forced to disclose the source of information or will be subservient to the government.
But enjoins enjoys a corresponding duty on the part of the Press—that it should maintain strictly honest journalism and should not give any garbled report or prejudicial account. Independent-minded editors should have the right to ft free expression of opinions and unhampered publication of news, subject only to the limitation that they do not publish abuse and keep within the bounds of decorum.
But real freedom of the press can exist only where a free people can function freely as in true democracy. It may be asked, why do we say there is no freedom of the press in England or America or India.
The reason is that in these countries, the common people are in economic bondage. Governments are ruled by the power of money and the press is controlled by owners in the interests of Big Business.
In is, therefore, rightly claimed that in a Socialist country as the people become economically free, the government acquires an increasingly representative character. The journalists then can write freely and fearlessly. The best way to ensure freedom of the press is to publish all news from accredited sources, to allow free ventilation of opinion, to do away with all controls except in the interest of public safety or welfare.
Freedom of the press should be a valued privilege and has to be safeguarded. It is a sacred right, which should be zealously promoted. Governments owe it to themselves to guarantee it from all arbitrary interference. Editors owe it to the public to maintain an honest and fearless attitude in favour of the people, against party or class interests.
And the people owe it to their country to demand and defend such freedom as a priceless heritage.But freedom of the press in each case depends on the way it reflects the will, the purpose of the people as a whole, in preference to that of any class or community or individual.
The illusory or unrealistic character of the freedom of . April , rev. April (This article is derived from a talk given at the Franz Developer Symposium.) In the summer of , my friend Robert Morris and I started a startup called torosgazete.com plan was to write software that would let end users build online stores.
"The Engineering of Consent" is an essay by Edward Bernays first published in , and a book he published in Philosophical Inquiries into the Nature of Human Freedom [F.W.J.
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Have no time nor desire to write your college papers? Apply for essay help at our legit, professional writing service. Have your paper written by the best expert in . Freedom Movement Bibliography. See also: Books Written by Freedom Movement Veterans Book Titles Grouped by Subject Film, Videos & Audio Movement-Related Web Links.


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In Beacon Heights, a seemingly perfect town, a group of three college friends struggle with the stress of being overachievers. In the aftermath of the town's first murder, each Perfectionist hides behind a secret, a lie and an alibi.
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Young CIA analyst Joe Turner has his idealism tested when he learns that the CIA has been using an algorithm he developed to spy on American citizens, leading the organization to a terrorist plot that threatens the lives of millions. Inspired by Sydney Pollack's 1975 political thriller Three Days of the Condor.
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Perhaps their strikingly different personalities make the relationship between detective Jane Rizzoli and medical examiner Maura Isles so effective. Jane, the only female cop in Boston's homicide division, is tough, relentless and rarely lets her guard down, while the impeccably dressed Maura displays a sometimes icy temperament — she is, after all, more comfortable among the dead than the living. Together, the best friends have forged a quirky and supportive relationship; they drop the protective shield in each other's company, and combine their expertise to solve Boston's most complex cases.
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Question about Toro Garden
The cover looks like it should just snap loose, maybe with a screwdriver, but I don't want to break anything. I was mowing when the self-propulsion feature suddenly quit woking. The drive belt does not appear to be broken, but is loose and the drive mechanism will not engage. Suggestions will be appreciated.
Posted by tipecanoe_20 on
It is a Toro 22" Recylcer
I have a 2008 Toro 22 in recycler and the self propel quit working and the rear wheels will not turn backwards. I tried tightening the cable as described in the manual, but that did not help. I am curious about how to remove the cover for the drive belt.
Thanks for your comments. The first thing I checked was to see if the drive shaft was clean, and it was much cleaner than I expected.Is the screw on the top of the deck or below the deck? I have not found it yet. Update: I think I just found it below the deck near the front.- Thanks again for your help.
hello mr t, is this mower a toro or a sears.
On my Toro recycler, I also had the front drive stop working -- and fixed it after reading the solutions here.
First, the plastic cover on the top deck that goes over the front drive mechanism must be loosened enough to pull it up. Before you can do that, on the underside there is one screw to take out.
Once that's done, all you have to do is pry the plastic cover up enough to see the belt and pulley that make up the front drive assembly. On mine, the belt had simply come off the pulley. Putting the belt back on, then tightening things back up is all it took to put the Toro back in sel-propelled happiness. Good luck!
Posted on Sep 05, 2010
I just found them. One at the left corner of the cover (standing facing the front of the mower) the other to the left of the opening where the cable enters the cover. You access them both from under the mower.
Posted on Oct 20, 2012
There were TWO bolts/screws (10 mm; I used a 3" socket to remove); located underneath; one is near the front wheel, the other covered up with dried grass near the blade housing.
Posted on Sep 26, 2011
Hi there,
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Posted on Jan 02, 2017
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text: Chandigarh: The inability of the JJP to finalise the name of its MLA for Cabinet berth has led speculation about the alleged differences within the party. The non-inclusion of either Ram Kumar Gautam (Narnaud) or Ishwar Singh (Guhla Cheeka) in the Cabinet from the JJP quota has reportedly not gone well with some of its MLAs. Reports said the BJP had reservations about Gautam, who defeated Capt Abhimanyu. TNS


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Refugee crisis "Turkey, Jordan and Lebanon are likely to falter"
The Obama administration ridiculed for not flying to the aid of its European allies, Turkish, Jordanian or Lebanese overwhelmed by the flood of refugees and migrants.
The United States is under pressure to lead an international emergency to the crisis of Syrian refugees, otherwise, are alarmed experts, to see collapse of the Middle East and the EU European.
The Obama administration is no longer vilified for months for refusing military intervention in Syria major, but is now ridiculed for not flying to the aid of its European allies, Turkish, Jordanian or Lebanese overwhelmed the flow of refugees and migrants.
Stressing that "the current refugee crisis is far from the worst since the end of World War II," the former ambassador of the United States in Iraq and Syria, Ryan Crocker, said that this potential influx of millions of displaced "is not a problem for the Middle east, or Europe, (but) a problem for the world and for America."
Worse, the former diplomat gives echo to the concerns of US officials that evoke recent weeks in a private threat "existential" on Europe and he fears too that "the flow of refugees to undo the European Union as a political construction" . As for the "frontline States, Turkey, Jordan and Lebanon, may falter," he warned. Indeed, King Abdullah II, the Jordanian kingdom welcomes hundreds of thousands of Syrians, had sounded the alarm to the breaking point.
So Mr. Crocker calls the US government to organize as soon as possible "a world summit on refugees" because "neither the region (the Middle East, ed), nor Europe can overcome" the crisis. But according to the US ambassador, the Obama administration has so far sin by "lack of leadership" while only America "can make the difference" to resolve the crisis.
"Global crisis"
Accused by his opponents of conducting a foreign policy "isolationist" towards the Arab world and Europe and not wanting to meddle in regional armed conflicts, the Democratic administration relentlessly defended its diplomatic and humanitarian commitment to Syria.
John Kerry even acknowledged Monday night for the first time, that the refugee crisis was a "global challenge" and not only "regional" for the Middle East and Europe. He admitted that he now was a "test for all of us" instead of "someone else's problem."
Faced with the Republican Congress, Mr. Kerry, who is sponsoring with Moscow a cease-fire and the delivery of humanitarian aid in Syria, had boasted last week that Washington was "the largest donor" for humanitarian Syria with "more than 5.1 billion dollars," released in five years.
President Obama also pledged last fall that the US welcomes 100,000 refugees of all nationalities by September 30, 10,000 Syrians. For the year 2016, they are for now just 942 Syrians have crossed all admission steps on American soil, according to official figures.
"You can not exercise leadership if you are not a leader," tackle Eric Schwartz, a former National Security Council of the White House and former head of the political department of state for refugees and migration.
The American Association Human Rights First makes the same basic criticism, stressing the "need for US leadership" in an murderous field report on the "deterioration of the situation of Syrian refugees." The author of the report, Eleanor Acer, denounced "the failure to resolve the refugee crisis (that) undermines the interests of national security of the United States, threatening the stability in the forefront of the country and contribute to the disunity of 'Europe".
It demands "the US government a welcome goal of 100,000 Syrians in 2017, a commitment that better reflects the tradition of American leadership and the interests of national security."
Moreover, agrees Mr. Schwartz, "this initiative will certainly encourage other countries to do more."
Translation of the article published on L'Orient-Le Jour website
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05/09/2018. Red Cross says with the crisis intensifying, an estimated 37,000 people were now crossing the Colombian border each day.
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27/04/2016. U.N. Resident and Humanitarian Coordinator Philippe Lazzarini chaired the briefing at the UNESCO office in Bir Hasan.


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The Role
As Team Leader, WFI Services you will be expected to provide day to day support and guidance to a team of Sales & Service Consultants. You will strive to provide optimal support of WFI Clients, colleagues and business partners.
Key Responsibilities:
Skills & Experience
About Us
At IAG, we believe that everyone has a unique point of view to share, shaped by their life experiences, cultures & passions. We celebrate and commit to:
#Proud to be me – we value difference, not sameness
#Together – harnessing our collective wisdom enables us to be our best for our customers & each other
#No boxes – it's not about labels, boxes or categories. It's about building a diverse and inclusive mindset into everything we do
IAG is the largest general insurance group in Australia and New Zealand. We own some of the region's most trusted brands, including NRMA Insurance, CGU, SGIO, SGIC and WFI.
Let's Talk
Start your career journey with us and click 'Apply'!
In addition to a diverse and inclusive culture, some of our benefits include 13% superannuation, 50% insurance discounts, flexible work and leave options, generous parental leave and return to work program, recognition and reward program, and various corporate partner discounts.
We encourage Aboriginal and Torres Strait Islanders to apply for this position.


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I was hoping to get some more changes in, but this appears to be more pressing. I am getting a loaner laptop that meets all but the OS specs listed above, so will need to dust off my XP install cd and get my hands dirty this weekend. You likely did not install the SATA drivers. If you could attach that here, it would be most helpful. So, listed in my Programs and features are the following Dell related progs.
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You need to log in to change this bug's status. There is a program at http: Read the documentation on the site, but I believe you only need to open a cmd window and run "dskwipe -l" to get a listing.
Download driver for laptop, Dell Computer Corporation Latitude E6400
Bug attachments screen when error happened edit Add attachment. The fact that it lists your SD card as removable, yet Win32ImageWriter won't detect it is very puzzling. Only took all day just to get to this stage. Controllrr questions Image Writer: Digital media reader v 4-in-1 digital media reader slot on some models Thanks.
I'm going to try running it in compatibility mode for Vista. Can either of you include info on what system you are running on? Malcolm mal wrote on Do you already have an account?
DskWipe – l output: Yes, my password is: This fails for me. Sorry, I was not able to get this resolved before releasing v0. Under device manager if found: I'll look into writing up a short test program in the next few weeks.
networking – Wireless internet not working: unclaimed – Ask Ubuntu
If you could attach that here, it would be most helpful. Please be patient, and thank you for your support.
JefferiesDec 26, at 7: I got the Nvidia card and only get a score of 3. Jeff B skydiver38 wrote on Duplicates of this bug Bug Bug Bug Bug Email me about changes to this bug report.
I have the same issue with the Dell ControlPoint. If I get time, I'll take that program and remove the potentially dangerous stuff, or just add a -d option to our tool to generate a debug log I personally like this idea best – but it will take more time. I'm a bit confused about that. Thank you all for your patience and support.
Fix will come very soon. None listed in the specs you linked to. You likely did not install the SATA drivers.
Windows 7 on Latitude E6400
I have a Thinkpad R61 with the same issue. Log in or Sign up. There are also entries for – Integrated Webcam Driver.


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Product News
Smart Thermal camera has explosive performance in GIT Security Award 2020
Hikvision, the world's leading supplier of innovative video security products and solutions, has won a GIT Security Award for its Thermal Explosion Proof Camera (DS-2TD2466T-25X). In a vote by GIT's security professional readers, the product was hailed as winner in the 'Fire and Explosion Protection' category of the popular annual awards. One focus of the…Read More
Hikvision showcased its latest innovative AI Cloud practices at CPSE 2019
Hikvision, the world's leading provider of innovative video security products and solutions, exhibited its latest innovative technologies, products and solutions at the China Public Security Expo (CPSE) 2019 in Shenzhen from October 28 to 31. This year marked the second anniversary of Hikvision's AI Cloud. Hikvision's presentation was dubbed "Fusing Data for a Smarter World"…Read More
The new SMARTair Lock puts advanced electronic access control into a familiar format
Now you can combine the advanced access control of a wireless electronic escutcheon with the intelligence and superior security of an electromechanical mortise lock With a robust design, the new SMARTair Lock is built to secure doors where high daily traffic and a large number of access events are all in a day's work. This…Read More
Ring door view cam, Ring's fifth video doorbell, now available for SAR 729
Perfect for apartments, flats, townhouses and rentals, Ring Door View Cam is a wire-free video doorbell that transforms a door viewer into a smart security device. Ring, whose mission is to make neighborhoods safer, today launched its fifth video doorbell, Ring Door View Cam in Saudi Arabia for SAR 729 (excluding VAT) at online retail…Read More
Tyco Engage Conference 2019
The 230 delegates who attended the recent Tyco Engage Conference 2019 were among the first to see how Tyco, the security products division of Johnson Controls, is continuing to help system integrators grow their businesses by developing innovative solutions which capitalise on emerging technologies. 'Beyond Security' was the central theme of the conference held at…Read More
Fingerprint-based drug screening test accelerates identification of drug mule body packers at airports
Simple and rapid fingerprint drug screen helps airport and border control teams to act on intelligence received on potential drug mule suspects within just 10 minutes Revolutionary technology screens for high-value opiates and cocaine typically carried by body packers A new fingerprint drug screening approach is now available to provide airport and border control staff…Read More
Managing Security Risks in The Oil and Gas Sector IQ Level 4 Course now Available Online
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New IDIS Micro Dome ideal for high end settings
The compact design of the DC-C4212RX allows for faster and more convenient installation and is the perfect choice for retail and discrete security applications. The new DC-C4212RX 2MP micro dome camera is the latest addition to the DirectIP® line up from IDIS, which is South Korea's largest in-country manufacturer of surveillance technology. The compact design…Read More
BIOS Middle East only regional player in Gartner's Magic Quadrant on DRaaS
For the 3rd consecutive year BIOS Middle East is recognised as the only regional player in Gartner's 2019 Magic Quadrant for Disaster Recovery as a Service (DRaaS). BIOS Middle East, UAE's leading Managed Cloud Provider, announced that it has been included in the latest Gartner Magic Quadrant report on Disaster Recovery as a Service (DRaaS),…Read More
IDIS launches AI in the box deep learning analytics
Plug-and-play AI to cost-effectively boost surveillance power and efficiency IDIS has launched its AI in the Box (DV-2116), boosting the power of surveillance systems with the most accurate deep learning analytics yet developed. Korea's largest surveillance manufacturer says in independent tests IDIS Deep Learning Analytics (IDLA) has achieved industry-beating accuracy rates of 97%, a record…Read More


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Coffee Monster! A fun new coffee app that allows you to skip the queue.
Entries Open: A Shot in the Dark 2020
Limited Edition Jonsson and Coffee Magazine Tees: Rise Up Africa!
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The Rise of the Seasonal Beverage with World Champion, Michalis Dimitrakopoulos
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Tell us about a coffee event in your area and we'll spread the word!
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Day Three: Bialetti Moka + Bialetti Venus
Day Two: Delter Press + Hario Mill Grinder from Equipment Cafe
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Coffee Club
We've been following Barista Hustle weekly and this latest post really struck a chord.
Friday, 19 June, 2015
I tasted some amazing coffees while on the West Coast of the US, but what really stood out were two facts:
1. American people can QUEUE! There were lines everywhere for everything. There was a line at the Ferry Building Market that was 400 people strong to pet a goat! Most cafes you walk into have a sizable queue before you get to the counter, which leads me to point #2...
2. American baristas are extraordinarily good at Head's Up service! I never felt uneasy or impatient in a queue, because the baristas behind the machine made me feel completely comfortable and taken care of. My order was important to them and I felt confident they were going to get to it as fast as they could. In the meantime their smile was a worth the wait.
This is an excerpt taken from the
Barista Hustle post
that reminded me just how important this skill is:
"It is your job as a barista to make sure that your service is flowing correctly, and to do this, you have to be watching. A service space is nothing without a flow, and especially not a busy cafe. Even if there is a line out the door, a quick bit of eye contact with each person shuffling in makes sure that the line is moving, gives you a feel for the speed of transactions and tickets, and helps build the crucial "we're all in this together" bonhomie of a relaxing service interaction."
South Africa has some way to go with this. Creating a flow and welcoming atmosphere within the cafe space is often not held at a top priority. Perhaps because there isn't that much competition of places to get a good cup of coffee? vida e caffe made their presence felt by the amazing service provided by their singing, smiling baristas, you couldn't help but leave that place in a good mood! With the mushrooming of this brand, this is one of the aspects I often miss most, not the coffee, but the experience.
A good barista can almost make a coffee with their eyes closed they've done it so many times, the next step to face those eyes forward and make the customer's experience of that cup of coffee world class. Acknowledging people's existence goes a long way towards a higher standard in cafes (and a healthy Tip Jar!).
Do you agree? Disagree? What are your best and worst cafe experience related to a head's up barista?
The Blue Bottle satellites all over San Francisco are always busy, but you never feel you've been waiting long. You have had to wait for your coffee, but the heads up service makes sure you never even notice!
Visiting one of the many wonderful markets, there are always queues of people waiting. Sightglass Coffee handled it like champs!
Got something to say? Then leave a comment!
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Kyrgyzstan Talk with Joan Pollock
11-12-2019 Doors open at 18:30. Talk starts at 19:00.
75 St Mary's Road, South Ealing W5 5RH
// 0.00
Join us at our London headquarters where writer, photographer and charity-founder Joan Pollock will be giving a talk about our Kyrgyzstan 'Art of the Nomads' group tour and the annual Shyrdak Festival of Felt Art.
If you have any burning questions about Kyrgyzstan, you will have the opportunity to ask them during the Q&A session followed by samples of traditional Kyrgyz cuisine.
Our team of experts will be on hand after the talk to share their wealth of knowledge and provide you with plenty of inspiration for your next holiday.
To reserve your place today Click here
Meet The Experts | Japan Travel Talk
26-11-2019 Doors open at 18:30. Talk starts at 19:00.
75 St Mary's Road, South Ealing W5 5RH
// 0.00
Join us for an evening of sushi and sake and find out more about the Nakasendo Way and visiting Japan. The evening will include a talk given by our team of Japan experts and a Q&A session with the opportunity to ask us any burning questions you have about this beautiful country.
To reserve your place today Click here.
TransIndus Summer Party
06-07-2019 12:30
48 Corringway, Ealing, W5 3AD
// 0.00
We took great pleasure in inviting our clients to the TransIndus annual Summer Party on Saturday 6th July 2019 at my home in Ealing.
This was a great opportunity for us to hear all about adventures from around the globe, for us to share some of the exciting things we have been up to and thank our past travellers personally for the support they have given us over the years for choosing to travel with TransIndus. It was a relaxed afternoon over drinks and lunch and we look forward to inviting you to our future events.
Transindus Exclusive Talks
30-05-2019 18:30 to 21:00
Transindus Offices at 75 St. Mary's Road, London W5 5RH
// Complimentary
We were delighted to announce the latest in our series of talks at our office which took place in South Ealing on 30th May.
Those of you who regularly attend our travel soirées at the Old Fire Station in South Ealing will already know David Abram, author of the Rough Guide to India, who has given a few memorable talks for us in the past.
This time he was sharing with us insights, anecdotes and top tips gleaned from various explorations of the Malabar Coast, in the far north of Kerala; an area he covered in detail for his best-selling Rough Guide to Kerala, and which he rates above all others in this part of the world.
"When people ask me which areas of the state they should visit, I always recommend the far north, because it has the same compelling mix of backwaters, beaches and mountains, as well as some unique cultural experiences, but with a fraction of the number of visitors. It's the home of Teyyem rituals, the beach where Vasco da Gama first landed in India and some wonderful forest hideaways."
Thank you for joining David and the TransIndus team for an evening of sociable, entertaining travel chat about hidden Kerala and other undiscovered regions of Asia. We laid on our usual complimentary South Indian buffet and we enjoyed hearing about your recent adventures, as well as, hopefully, providing you with inspiration for future journeys!
Cities of the Medieval Silk Road
17-11-2017 Doors open at 18:30. Talk starts at 19:00.
The Embassy of the Republic of Uzbekistan, 41 Holland Park, London W11 3RP
39329694174 // Free
An illustrated talk as we explored the cities of the medieval Silk Road with Dr Paul Wordsworth, an expert on Islamic archaeology and ancient Central Asian trade routes.
Before the opening of the sea route around the Horn of Africa in the 16th century, the principal way valuable merchandise travelled between China, India and Europe was overland, via the vast deserts and mountain ranges of Central Asia. Silk, a fibre whose origins were kept a closely guarded secret for thousands of years by the Chinese, formed the mainstay of this ancient trade, which is why the tangle of tracks along which it travelled came to be known as the 'Silk Road'.
Writers from Goethe to Keats were fascinated by the near-mythic cities that punctuated the trade arteries: Yarkhand, Kashgar, Bishkek, Khiva, Samarkand and Bukhara... Their very names evoke images of great caravans of twin-humped Bactrian camels marching across vast, undulating steppes, of turquoise-tiled minarets and of fabulous bazaars.
Dr Wordsworth is a Research Fellow at the University of Oxford, focusing on the archaeology of the medieval Caucasus and Central Asia.
Michael Wood: Dynasties of the South
05-10-2017 Doors open at 17:00. Talk starts at 17:30.
The Nehru Centre, 8 South Audley Street, London W1K 1HF
35258576351 // Free
An illustrated talk by historian and broadcaster Michael Wood was hosted at The Nehru Centre in London.
Michael has acquired bona fide 'national treasure' status in the course of his long and prolific career, which began with his landmark series on The Dark Ages in 1979 and has included several notable forays into Asia.
His books and television programmes on India are perfect primers if you plan to travel to the east. We routinely recommend his brilliant summary of the roots of Indian civilisations in Legacy (1992), and The Smile of Murugan (1995) is an absolute must for all travellers to Tamil Nadu.
Anyone who has had the pleasure of attending one of his lectures will confirm that Michael's ability to bring past eras to life is second to none and, if anything, even more compelling in front of a live audience than on the small screen.
In this lecture Michael took us on a journey through South India, exploring its cultures, temples, towns and ritual traditions.