Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site–like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5′ to 3′ specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2–independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes. Proteins have been identified for which a unique translational machinery makes use of unconventional start codons Immune surveillance by cytotoxic T cells (CTLs) is a key mechanism for detecting and eliminating abnormal cells. These include cells infected with viruses or bacteria, and those that have suffered tumorigenic transformations . The antThe antigen-presenting cells (APCs), which include almost all nucleated cells, are also very efficient in generating peptides for display by MHC class I . In factiMet, which is specific for AUG and is always charged with the methionine residue -TFNYRNL peptide (the initiation codon is in parentheses and the remaining amino acids are in single-letter code) . AlthougRNAiMet . In the d by CAA . Becaused by CAA . TherefoTo address this question, we identified the first amino acid of CUG-initiated peptides that were presented by MHC class I molecules and thus detectable by appropriate T cells. In this assay, we cotransfected cells with cDNA constructs encoding the peptide as well as the MHC molecule that binds and presents the peptide on the cell surface. Binding to the MHC protects the initiation residue from being removed by cellular proteases, which can trim antigenic peptides in the cytoplasm or the endoplasmic reticulum . We thenb MHC molecule and the *(CTG)-TFNYRNL* peptide ([CTG]YL8). We refer to this as the xYL8 model. We placed the peptide in three different contexts: first, in the pcDNA1 vector; second, upstream of the IRES in the pIRES2-eGFP vector; and, finally, in the 5′ UTR of green fluorescent protein (GFP) in the pcDNA1 vector. The peptides translated in the transfected cells were extracted and fractionated by HPLC. Each fraction was tested for the presence of the leucine-initiated LTFNYRNL (LYL8) peptide and the methionine-initiated MTFNYRNL (MYL8) peptide using BCZ103 T cells and Kb-expressing L cells (a fibroblast cell line) as APCs. With each construct we found a single peak of antigenic activity that eluted in the same fraction as the synthetic LYL8 peptide (To determine if decoding of the CUG initiation codon as leucine was influenced by 5′ or 3′ untranslated regions (UTRs) of the mRNA, we transfected COS-7 cells with cDNA encoding the K peptide A–1C. In  peptide A and 1D.b MHC class I molecule that satisfied the conditions required for our assay: that an MHC molecule present the peptide, that HPLC allow distinction between the leucine- and methionine-initiated forms, and that a T cell cross-react with the peptides with leucine or methionine residues at the first position. When COS-7 cells were transfected with cDNA constructs encoding the Db MHC molecule and the *(CTG)-SNEN-METM peptide derived from the influenza nucleoprotein, only the leucine-initiated LM9 peptide was detected in the HPLC-fractionated cell extracts  and read through the stop codon before the CTG initiation codon in the LYL8 coding sequence. We showed that increasing the number of stop codons upstream of CTG from one to six had no effect on LYL8 expression, and that substituting the CTG codon with other leucine-encoding triplets essentially ablated peptide expression . We asse complex B and the complex C. T cell complex C. To con complex D. Again,We also considered the possibility that leucine was used as the first amino acid because of an RNA modification that introduced an AUG immediately upstream of the peptide-coding sequence. However, the experiments below show that the 5′ UTR influences the leucine start because it is affected by the Kozak context, by the presence of an upstream hairpin, and by the presence of upstream initiation codons. Together, these findings demonstrate that the mRNA remained intact.GCCACCAUGG is optimal, and that the nucleotides at positions –3 and +4 are the most influential, while the nucleotide at the –6 position exerts a smaller effect. Changing these nucleotides can change protein expression by 20-fold SEL8* into the pcDNAI vector, where N represents the degenerate nucleotides flanking the CTG initiation codon for the SLVELTSL (SEL8) peptide presented by the Kb MHC molecule to bm1BZ19.4 T cells SEL8* stimulated the T cells, demonstrating that initiation activity was restricted to the CTG codon  for the AUG start and extends earlier findings that showed that an A at +5 and U at +6 can also enhance initiation at the CUG codon YL8 model, in which the predominant T cell-stimulating activity is the leucine-initiated LYL8 peptide see A–1C. At b MHC cDNA. After 2 d the transfected cells were assayed for their ability to stimulate the BCZ103 T cell. Cells expressing the LYL8 peptide with its CTG initiation codon in the “Excellent Kozak” context  were superior to those with the CTG codon in a “Poor Kozak” context  in stimulating the T cell response  T cells A and 6B. T cells .3CTG. When the transfected cells were used directly to stimulate T cells, the upstream ATGs had little effect (We then transfected cells with a construct encoding (CTG)YL8 and another encoding (CTG)YL8 with three ATGs upstream of and out of frame with the peptide (ATG)e effect C. Howevee effect D. Thus, 3CTG. When the transfected cells were used directly to stimulate T cells, we saw a small but consistent inhibition YL8 with three CTGs upstream of and out of frame with the peptide (CTG)hibition E. Remarkhibition F. The obNote, however, that the upstream CUGs, despite an “Excellent Kozak” context, inhibited the leucine start weakly. This effect contrasts with the inhibition caused by the upstream AUGs on the AUG/methionine or the CUG/methionine starts and suggests that other features are required for an efficient CUG/leucine start. Interestingly, one form of the ASCT2 amino acid transporter is initiated with multiple CUG and GUG codons in close proximity . This reiMet onto the 40S ribosome. Cells target eIF2 by phosphorylating its α subunit (eIF2α) to inhibit protein synthesis in response to a number of stress signals, including viral infection, starvation, and the accumulation of unfolded proteins. Ribosomes release eIF2-guanosine diphosphate (GDP) after the AUG initiation codon is reached, and GDP is exchanged for guanosine triphosphate (GTP) with the assistance of another protein, eIF2B, before eIF2 can be used for another round of translation initiation. When eIF2α is phosphorylated, it binds eIF2B with unusually high affinity and thus prevents subsequent nucleotide exchange. Because eIF2B is limiting in the cell, phosphorylation of only a fraction of eIF2α can substantially inhibit translation globally , which is responsible for loading the RNAglobally .To approach this question, we transfected HeLa cells with (CTG)YL8 or (ATG)YL8 constructs. We then assayed peptide expression in cells that had or had not been treated to induce phosphorylation of eIF2α. It was a challenge to induce phosphorylation of eIF2α for long enough to see an effect on peptide expression without causing substantial toxicity. Furthermore, we could not disrupt peptide/MHC assembly in the endoplasmic reticulum, a requirement that ruled out standard reagents such as dithiothreitol, thapsigargin, and tunicamycin. We also did not want to inhibit peptide elongation, which ruled out amino acid starvation.The optimal treatment for our purposes was sodium arsenite (NaAs). Arsenite reacts with sulfhydryl groups and causes phosphorylation of eIF2α presumably by inducing an unfolded protein response, although the precise mechanism remains unknown . We tranWe assayed the HeLa cells for peptide expression using the BCZ103 T cell hybridoma B. We fouThe effect of eIF2α phosphorylation on the leucine start strikingly mirrors the effect of eIF2α phosphorylation on proteins whose synthesis is directed by the CPV-IRES, which does not require eIF2 . IntriguThe CPV-IRESs are to date the only known sequences that allow eIF2-independent initiation in eukaryotic cells. Viruses employ a host of creative strategies to prevent phosphorylation of eIF2α . InitiatIn summary, we found that when CUG acts as an alternate initiation codon, it can be decoded as leucine as well as methionine. The leucine start does not depend on mRNA structure or sequence, but its efficiency can be enhanced by the Kozak context. A set of ribosomes is scanning 5′ to 3′ specifically for the CUG initiation codon. While the methionine start is inhibited when cells are treated with NaAs, the leucine start is enhanced, suggesting that leucine initiation is independent of eIF2. This novel translation initiation mechanism provides cells not only antigenic peptides but also a potential tool for translational control.–, COS-7, Kb+B7.2+ L, Db+B7.2+ L, BCZ103, 30NX/B10Z, and DBFZ cells have been described . HeLa cells were obtained from ATCC , and cultured in Eagle's minimal essential medium modified with Earle's balanced salt solution, nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1500 mg/l sodium bicarbonate (ATCC #30–2003), 10% fetal bovine serum, and 10 μg/ml ciprofloxacin.Lmtkescribed . Cell liSequences for constructs depicted in Sequences for constructs depicted in Sequences for constructs depicted in Sequences for constructs depicted in Sequences for constructs depicted in 3ATG-YL8 in the BstXI/XbaI sites of the pcDNA1 vector,5′-TGTGAACCCAGGGTCGACCCAGGACCCAGGTAGTCGACCATGACCTTCAACTACCGGAATCTCTAG-3′; and ATG3ATG-YL8 in the BstXI/XbaI sites of the pcDNA1 vector,5′-TGTGAACCATGGGTCGACCATGGACCATGGTAGTCGACCATGACCTTCAACTACCGGAATCTCTAG-3′.Sequences for constructs depicted in 3CTG-YL8 in the BstXI/XbaI sites of the pcDNA1 vector,5′-TGTGAACCCAGGGTCGACCCAGGACCCAGGTAGTCGACCCTGACCTTCAACTACCGGAATCTCTAG-3′; and ATG3CTG-YL8 in the BstXI/XbaI sites of the pcDNA1 vector,5′-TGTGAACCATGGGTCGACCATGCACCATGGTAGTCGACCCTGACCTTCAACTACCGGAATCTCTAG-3′.Sequences for constructs depicted in 3CTG-YL8 in the HindIII/XbaI sites of the pcDNA1 vector mutated to alter the two CTGs in the 5′ UTR to CAG,5′-AGCTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTTAGAAACTGACCTTCAACTACCGGAATCTCTAG-3′; and CTG3CTG-YL8 in the HindIII/XbaI sites of the pcDNA1 vector mutated to alter the two CTGs in the 5′ UTR to CAG,5′-AGCTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACTGGATTACAAACTGGATTACAAACTGGATTTAGAAACTGACCTTCAACTACCGGAATCTCTAG-3′Sequences for constructs depicted in 3CTG-YL8 in the HindIII/XbaI sites of the pcDNA1 vector,5′-AGCTACAAACAGGNTTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTTAGAAACTGACCTTCAACTACCGGAATCTCTAG-3′; and CTG3CTG-YL8 in the HindIII/XbaI sites of the pcDNA1 vector,5′-AGCTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACAGGATTACAAACTGGATTACAAACTGGATTACAAACTGGATTTAGAAACTGACCTTCAACTACCGGAATCTCTAG-3′.Sequences for constructs depicted in Sequences for constructs depicted in – or COS-7 cells were transfected in 96-well plates (1 × 104 cells per well) with the indicated concentration of plasmid DNA, 0.1 mg/ml DEAE-dextran, 0.1 mM chloroquine, 10 ng/ml MHC class I cDNA, 5 ng/ml B7.2 cDNA, and 10% NuSerum  in RPMI 1640. After 90 min, the cells were shocked with 10% DMSO in PBS for 2 min. After 2 d incubation in normal medium, T cells (1 × 105 cells per well) were added to assay peptide expression. GeneJuice  transfection reagent was used in The DEAE-dextran transfection method was used in 5 cells) were incubated with APCs in 96-well plates for 6–24 h. The response, accumulation of intracellular β-galactosidase, was measured with the substrate chlorophenol red-β-D-galactopyranoside (CPRG). The product was measured with a 96-well plate reader at 595 nm and 655 nm as the reference wavelength.The T cell assay has been described previously . Briefly4 per well) and T cell hybridomas (1 × 105 cells per well).The HPLC assay has been described previously . BrieflyCells were transfected with GeneJuice (Novagen) as described above, but the transfection medium was left on for only 4 h before the cells were lifted and split into multiple dishes with fresh medium. Cells were allowed to rest for 12 h before sodium arsenite treatment. They were treated with 50 μM NaAs , 1× GolgiPlug containing brefeldin A , or left untreated for 4 h. The cells were then lifted and counted. For the T cell assay, they were titrated in a 96-well plate. The assay was as described above, except that 1× GolgiPlug was added in order to “freeze” the cells in their state at the end of treatment. For the Western blot, they were incubated on ice for 5–10 min in lysis buffer . The lysate was spun for 15 min at 4 °C, and the supernatant was transferred to a tube containing an equal volume of 2× SDS-PAGE sample buffer . The sample was then heated in water just off the boil for 5 min, separated on a 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature in TBS–0.1% Tween 20–5% bovine serum albumin , incubated for 1 h with primary antibody  in TBS–0.1% Tween 20–5% bovine serum albumin, washed four times for 5 min with TBS–0.1% Tween 20, incubated for 40 min with secondary antibody , washed four times for 5 min with TBS–0.1% Tween 20, incubated for 5 min in substrate , and exposed to film. An antibody to α-tubulin  was used as a loading control.


In the spirit of good health, cells are constantly subjecting their protein contents to immunological surveillance by cytotoxic (killer) T cells. Tens of thousands of major histocompatibility complex (MHC) class I molecules cradle peptides (bits of proteins) on cell surfaces, and T cells detect any suspicious peptides with extreme sensitivity. If a cell is infected with a virus, peptides created from viral DNA will end up on the cell's surface as antigens, triggering immunological red flags.Most—but not all—of the peptides presented by MHC class I molecules are created by conventional cellular mechanisms: with the help of a ribosome, three mRNA nucleotides (a codon) are decoded into a corresponding amino acid, which is strung as the next link on an elongating peptide. Most peptides begin with the amino acid methionine, coded by the mRNA nucleotide triplet A-U-G (AUG). But some peptides are “cryptic,” arising from normally untranslated regions of mRNA or initiated with codons other than AUG.not begin with methionine.Previous studies suggested that an unconventional translation mechanism creates some cryptic peptides. But how? And why? Only one type of translation initiator, transfer RNA (tRNA), specific for AUG and loaded with a methionine molecule, is known. Protein synthesis beginning at alternate codons has been attributed to imprecise pairing between the methionine translation initiator and mRNA. This, however, does not explain proteins that do Only two mechanisms for building non-methionine-initiated peptides have been discovered. In a new study, Susan R. Schwab et al. characterize one of them, the CUG-initiated translation of a peptide starting with leucine instead of methionine.The authors explored cellular translation by engineering cells to create peptides of interest and present them through matching MHC molecules on the cells' surfaces. Then, by harnessing the exquisite sensitivity of T cells to probe for antigens on MHC molecules, they could identify which peptides were created under different experimental conditions.Their findings point to a unique translation mechanism. In the other known example of a non-methionine-initiated peptide, translation beginning at GCU or CAA is guided by a specific folded structure of mRNA nucleotides called the internal ribosome entry site. Schwab et al. have found that no similar structure is necessary for CUG-initiated translation. However, similar to the standard mechanism of AUG initiation, they found that ribosomes do scan for CUG. Additionally, the presence of a specific ribosome-binding sequence in mRNA (the “Kozak context”) near a CUG site can enhance the efficiency of initiation there.Schwab et al. have also suggested a possible purpose for this translation mechanism. Under stress, cells can down-regulate conventional translation, which curbs the production of viral proteins in the event of an infection but also suppresses the creation of antigens needed to flag down T cells for an immune response. Here, Schwab et al. report that peptides starting with leucine were produced in the absence of the protein eIF2, which normally aids in AUG-initiated peptide synthesis. Cells under stress slow conventional translation by restraining the function of eIF2. Therefore, CUG-initiated translation, which works without eIF2, might provide an out for stressed cells needing to create peptides. This alternative could be a great way to avoid pumping out viral proteins and still create antigens for T cell surveillance—unless, of course, viruses take advantage of the loophole for their own peptide production.


Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes. It is generally accepted that a normal cell has to acquire multiple mutations in order to become a malignant tumor cell. Considerable effort has been invested in finding single genes involved in tumor initiation and progression, but relatively little is known about the constellations of cancer genes that effectively collaborate in oncogenesis. In this study we focus on the identification of co-occurring DNA copy number alterations  in a series of tumor samples. We describe an analysis method to identify DNA copy number mutations that specifically occur together by examining every possible pair of positions on the genome. We analyze a dataset of hematopoietic tumor cell lines, in which we define a network of specific DNA copy number mutations. The regions in this network contain several well-studied cancer related genes. Upon further investigation we find that the regions of DNA copy number alteration also contain large networks of functionally related genes that have not previously been linked to cancer formation. This might illuminate a novel role for these recurrent DNA copy number mutations in hematopoietic malignancies. PIK3CA and KRAS genes was observed Tumor development is generally thought to be a process in which healthy cells transform into malignant tumor cells through the step-wise acquisition of oncogenic alterations Besides single basepair mutations or retroviral integrations, the activity of genes can also be perturbed by DNA copy number alterations that arise as a result of genomic instability, which is frequently observed in tumor cells n) DNA is performed on oligonucleotide- or Bacterial Artificial Chromosome (BAC) based microarray platforms. For each probe on the microarray, the ratio of signal intensities of tumor versus normal DNA is a measure of the relative DNA copy number of the corresponding genomic region in the tumor sample. Platforms designed to identify single nucleotide polymorphisms (SNPs) can also infer CNAs by comparing the raw probe intensity values measured in a tumor sample with a reference sample.DNA copy number alterations (CNAs) may be measured on microarray platforms In order to extract those DNA copy number aberrations that preferentially occur together, we developed an analysis framework. The basic premise of our analysis is to define a pair-wise score for any given pair of genomic locations present in the dataset. This scoring index will only be high if both genomic locations are recurrently aberrated in multiple independent samples within the tumor panel, and if they co-vary similarly over the different samples . Using aThe raw data consist of non-discrete measurements of the average DNA copy number of the population of cells present in the measured sample. The signal consists of a measurement of a heterogeneous population of tumor cells, which may contain many populations potentially carrying different mutations and copy number alterations, as well as normal (diploid) cells. To reduce heterogeneity as much as possible we choose to analyze a collection of hematopoietic tumor cell lines, which on a per-sample basis can be considered clonal. There were several other reasons for analyzing this particular dataset. First, it is a high resolution dataset of well-characterized, clinically relevant samples. Although these samples are cell lines, they are widely used as a model system for the diseases from which they have been derived. Second, this collection of samples includes cell lines derived from T- and B-cell leukemias carrying rearranged T-cell receptor and immunoglobulin loci, respectively. We therefore should be able to separate these two distinct lymphoid malignancies based on co-occurring DNA copy number losses at the T-cell receptor and immunoglobulin loci. During T- and B-cell development, these loci undergo DNA recombination and gene deletion in a process known as V(D)J-recombination. The human genome contains three specific T-cell receptor loci  on two different chromosomes that determine their variability. B-cells have three different loci  on three different chromosomes that undergo recombination to generate a diverse repertoire of immunoglobulins. Since T- and B-cells only undergo recombination of their respective loci after lineage commitment, it is unlikely that T-cell receptor loci are recombined in B-cells and vice-versa. If our approach is successful at finding co-occurring losses, it should identify the co-occurring rearrangements at the T-cell receptor alpha/delta and beta/gamma loci in T-cell leukemias. Similarly, we should be able to pick up co-occurring losses at the IgG kappa, lambda and heavy chain loci in B-cell malignancies.A classic example of finding associations in a large (binary) dataset is association rule mining. Identification of cooperating events in continuous data requires a different approach than binary association rule mining. First we developed a method to score for co-occurrence between two continuous measurements . We thenDNA copy number measurements at two different genomic loci can be visualized in a 2D space, with each axis representing measurements at a certain genomic locus. A point in this space represents a sample in which both loci were measured. We use the covariance of the two measurements to score for co-occurring loci. This score only rewards a high value to a truly co-occurring and co-varying pairs of measurements .For thisσ parameter of the Gaussian function used to convolve the score matrix  we expected to retrieve these co-occurring losses in the small (2 Mb) scale analyses. Since we disregarded co-occurrences on the same chromosome we expected to find five co-occurring losses. Indeed, four of the five expected co-occurring losses are present in the top 50 peaks of the Scale 2 analyses . Figure While the recovery of the V(D)J-related recombination loci as co-occurring losses serves as a positive control for our analysis approach, we are mainly interested in identifying cooperating genes or regions that might play a role in cancer. To see whether the locations we recover are linked to this disease, we analyzed whether the co-occurring genomic loci are enriched for genes known to play a role in cancer. As a reference gene set we used the Cancer Gene Census list While finding enrichment for cancer genes is an encouraging result, this does not explain the possible cooperation between two loci. We expect that the co-occurring loss of two regions points to a functional relationship between the constituents of the genomic loci. A co-occurrence between two genomic regions can point to many different kinds of interactions between the genes residing in both regions, e.g. biochemical interactions of the protein products or functional collaboration of two cancer genes in tumorigenesis. We therefore decided to employ interaction data to shed further light on the genes present in the co-occurring regions. We translated the co-occurring pairs of genomic loci to pairs of gene sets, and we investigated the functional relationships of their protein products using the STRING database In order to keep control of the complexity, we considered in our co-occurrence analysis only radially symmetric kernels, i.e. Gaussian kernels with diagonal, equal variance covariance matrices. This implies that asymmetric co-occurring regions – where a small locus co-occurs with a large locus – will not be optimally detected. Since an asymmetric co-occurring region typically consists of a series of symmetric co-occurring regions detected on a smaller scale (just like a rectangle can be constructed from a collection of squares), we set out to construct larger co-occurring regions from the results of the smaller scales using a hierarchical clustering approach. For details see Supplemental 8). This resulted in a 1000×1000 distance matrix. On this distance matrix we performed single linkage hierarchical clustering. The resulting dendrogram was cut at 1 * 107 bp (5 kernel widths). The resulting clusters are unique genomic loci and were represented as nodes in a graph. Clusters were then linked if a co-occurrence was found between individual loci of different clusters. These links are represented as edges in a graph. The result of the clustering analysis is shown in Briefly, we collected the loci involved in the top 500 co-occurrences of the Scale 2 analysis. This resulted in 1000 genomic loci. For each pair of loci, we calculated the genomic distance in base pairs. The distance between two loci on different chromosome arms was set to a default high value  , the MAPK pathway suppressor GPS2, the nuclear co-activator (and known tumor-suppressor) p300 and a gene of unknown function, CBFA2T3. All interactions found are based on physical binding and co-occurrence in Pubmed abstracts.We investigated the remaining 113 interactors for any interesting interactions that might be a target of this collection of co-occurring losses. Within the total network of interactors we found a sub-network centered on the nuclear co-repressor  (TRAC1) . This inTrp53 or p19-ARF deficient mice Cbfa2t3 is shown in Cbfa2t3 was not flagged as a common integration site, several viral integrations near this gene were found. Remarkably, two individual tumors harbored a bi-allelic integration near the transcription start site of Cbfa2t3, suggesting functional inactivation of this candidate tumor suppressor gene. Indeed, bi-allelic integration is thought to be a hallmark of tumor suppressor genes in IM screens To see whether we could find more information regarding the putative tumor suppressor function of the different interactors, we tested if we could corroborate our findings with data from a large retroviral insertional mutagenesis (IM) screen where hematopoietic tumors were induced through Murine Leukemia virus (MuLV) infection of wild-type mice or Given that we find this sub-network of interactors in a co-occurring network of DNA copy number losses and the recovery of inactivating insertions in a retroviral IM screen, we conclude that this network might be a putative tumor suppressor network.In this study we present a genome-wide analysis for finding collaborating DNA copy number changes on different chromosomes. Using our 2D kernel convolution framework we can score and find co-occurring DNA copy number changes in a high quality, high resolution aCGH dataset. Using a dataset of hematological cell lines we are able to recover DNA copy number alterations specific to the cell lineage of the samples. Furthermore we uncover cancer-related networks of co-occurring DNA copy number changes.Several studies have investigated concerted copy number changes in aCGH data. In studies on lung cancer n results, residing in the extremes of the results distribution, thus minimizing the chance of including false positives. The top n lists allowed us to generate workable results which we have validated extensively with other sources of evidence.The output of our analysis does not include a measure of significance. Constructing a background distribution based on permutations of the DNA copy number data would mean re-running our analysis thousands of times, a task which remains computationally infeasible at this stage. Furthermore, the multiple-testing problem would have to be properly addressed, given that the number of tests is the square of the number of grid points in the 2D space. Due to the complexity of the analysis procedure (minimum operation and kernel smoothing) the definition of an analytical null distribution has remained elusive. Therefore, we have chosen to work with top While we were able to use a distributed computing solution for our analysis, we were fortunate to have the required computational architecture at our disposal. Since the problem basically consists of repeating the same action many times it could be well-suited to software optimization or a hardware based solution where the most time-consuming actions are handled by a dedicated processing unit.When looking for areas in the 2D pair-wise space highly enriched for co-occurrence scores we convolve this space with a 2D-Gaussian kernel. The sigma parameter of this function is a representation of the size of the aberrations we expect to recover. Currently we make the implicit assumption that the co-occurring aberrations have the same size by using a symmetric kernel for the convolution. This could be relieved by allowing for an asymmetrical (ellipsoid) Gaussian kernel for all combinations of scales used. Clearly, this comes at the cost of increased computational complexity. Here we resolve this issue by concatenation of the results obtained in a small scale. In this way we can recover co-occurring losses of different sizes that give a better enrichment for functional interactions when combined with the single peaks obtained in a higher scale analysis.In our analysis of a set of cell lines derived from hematological malignancies we found enrichment of cancer related genes and functional interactions in co-occurring DNA copy number changes. Our results suggest that tumorigenesis requires elimination of multiple gatekeeper genes and gain of multiple oncogenes as demonstrated by the presence of many functional interactions between the loci in the gain-gain and loss-loss core networks.Haploinsufficiency is a well known characteristic of several tumor suppressor genes, where simple reduction of gene dosage by loss of gene copies at the DNA level can already promote oncogenic transformation TP53 gene, often thought to be the single target of this deletion TP53 is no doubt an important target of the DNA copy number loss, our analysis indicates that the concomitant loss of other genes near TP53, as well as co-occurring losses on the other genomic loci may together account for the full tumorigenic effect.We show that the 17p loss and its co-lost regions are highly enriched for functional relationships, which are not fully explained by the presence of the Loss of the loci on 17p, 9p, 9q, 13q, 16q and 22q has been reported previously for several types of hematological malignancies represented in our dataset TP53, RB1 and CDKN2A (INK4a/ARF). We found one sub-network of genes around NCOR1 which might be an example of other tumor suppressor genes that are affected by the concerted loss of these genomic loci.Not all of the gene-gene interactions defined by the 17p network involve the well-known canonical cancer genes NCOR1, is a well-known transcriptional co-repressor that associates in a ligand-independent manner with nuclear receptors SMRT, for recruitment of HDAC proteins to the DNA to induce transcriptional silencing. Its role in cancer is not well-established. NCOR1 null mice die in early embryogenesis NCOR1 is known to increase proliferation in hepatocytes NCOR1 decreases AKT phosphorylation, thus countering its pro-survival signal NCOR1 - might increase proliferation and promote survival.The hub of this interaction network, NCOR1 and its partner genes  have been based on co-occurrence in PubMed abstract and true physical binding CBFA2T3 is a close relative of ETO, which is a target of the recurrent AML1-ETO translocation that occurs in acute myeloid leukemia. It has been shown that the fusion gene AML1-ETO actually interferes with the CBFA2T3-NCOR1 interaction, and that its oncogenic effect derives from that inhibition Cbfa2t3 is recurrently targeted by bi-allelic retroviral integrations, which are predicted to cause functional inactivation of Cbfa2t3PPARA is a member of the Peroxisome proliferator-activated receptors, and has been implicated in hepatocellular carcinoma development in rodents PPARG, exhibit a tumor suppressor-like phenotype, it is possible that PPARA can act as a tumor suppressor in hematological malignancies. GPS2 is a known suppressor of JNK signaling MAP kinase signaling pathway. Deregulation of this pathway is a well-known phenomenon in cancer NCOR1 and the known tumor suppressor p300, our data suggest a selective advantage for loss of multiple constituents that interact with NCOR1 since they all may have tumor suppressor-like activities.All interactions between BRCA1/2-related breast cancer.Many studies focus on a single hematological malignancy in which a single combination of aberrations might be important NCOR1 that may be a novel network of tumor suppressor genes that are affected by the observed co-occurring losses. The observation that DNA copy number changes may affect gene dosage of larger numbers of cancer-relevant genes deviates from the classical view where mutations in a few (5–7) cancer genes lead to tumor development. Our data support the notion of cancer-related networks or pathways, where multiple collaborating genes are deregulated simultaneously to induce oncogenesis. Such a network view of oncogenesis is an important step towards developing effective drug targets because it increases the number of potential targets. However, this view also implies that multiple molecules need to be targeted simultaneously in order to achieve optimal therapy response and to reduce the risk of therapy resistance.We have developed a method for genome-wide analysis of collaborating DNA copy number changes and their corresponding networks. Using this approach we have identified a loss-loss network centered around a region on human chromosome 17p. This network is highly enriched for functional relationships and hints at a more complex system of tumor suppression in which many different genes are affected simultaneously to induce cancer. We show one example of a sub-network around the nuclear co-repressor Datasets consisting of array-based copy number measurements are continuously increasing in size. If probe level interactions are evaluated, the analysis space is of dimensionality Let the aCGH profile of the Negative and positive log2 values respectively denote loss or gain of DNA in the test sample versus the reference sample. We regard both situations separately, which prevents the negative and positive values cancelling each other through summation later in the algorithm. We separate gains and losses by only retaining grid positions with positive values for the gains or negative values for the losses. The absolute values of the separated matrices are then used in the downstream steps. The remaining grid positions are set to zero. More specifically, the gains matrix, The first component of the co-occurrence score is the continuous variant of the AND Boolean logic function: the minimum operation. For two grid points, Since we believe the co-occurrence score to be a smooth variable, and since neighboring co-occurrence values can therefore be employed to reduce the noise locally, we convolve the co-occurrence score matrix with an isotropic 2D Gaussian kernel function. In practice this implies sampling the 2D Gaussian kernel function on a square grid consisting of With 39 unique chromosome arms in the human genome (disregarding the p-arms of the acrocentric chromosomes and the sex chromosomes), three different scales and 4 triangular pair-wise matrices to evaluate  we compute 8892 different CCMs. To solve this problem computationally we used a large distributed computing cluster. Our choice of resolution of the genomic grid was bounded by the memory present on the nodes. We set th peak represents two co-occurring loci, For each CCM we determine the top N peaks for each combination of gains and losses. The nhttp://www.sanger.ac.uk/genetics/CGP/Census/). The reference list of all genes was retrieved from the Ensembl website, with a filter to keep only genes with bio-type = ‘protein_coding’. This left 18840 genes. All CGC genes that could not be mapped back to the reference gene set were excluded. The CGC genes that were annotated as ‘recessive’ were used as the tumor-suppressor genes and ‘dominant’ as oncogenes. Enrichment for all CGC genes, the tumor-suppressor subgroup and the oncogene subgroup in the gene sets determined by the co-occurrence analysis was calculated using a Fisher's exact test.The list of CGC genes was obtained from the CGC website . Clusters are represented as nodes in a graph. Edges between nodes are drawn if any co-occurrence relationship is found between loci present in the nodes.For all pairs of co-occurring loci, http://www.sanger.ac.uk/genetics/CGP/CellLines/). A list of the cell lines included in this dataset can be found in The case we subjected to analysis was a dataset containing 105 cell-lines derived from hematological origin. The aCGH measurements were done on 1.8 million probe Affymetrix SNP 6.0 arrays. After data pre-processing we were left with 95 samples. These cell lines are a subset of the Cancer Genome Project cancer cell line project Click here for additional data file.Dataset S1This file contains information about the cell lines used in this study.(0.03 MB XLS)Click here for additional data file.


It is not uncommon for a pharmaceutical company to develop several new drugs simultaneously for the same indication. In a small population it may be infeasible to try all new drugs concurrently. A practical approach is to consider one at a time and if the drug is recommended for a phase III trial to temporarily suspend development of other drugs.We propose a development plan that encompasses phase II and III trials, considering a series of sequential phase II trials with interim decision-making. At each stage of a phase II trial, a decision is made whether to continue with participant recruitment to the current trial, stop the current trial and recommend the drug to be tried in a phase III trial, stop the current trial and initiate a new phase II trial with a different drug or stop the current trial and abandon the development.The unknown treatment effect for each drug is assumed to follow a prior distribution. In addition, as drugs are intended for the same population it is not unrealistic to consider treatment effects to be correlated, so that the prior distribution will reflect this. As data are observed from preceding and current trials, prior densities for subsequent drugs are updated, informing us of the performance of the groups of drugs under evaluation. The application of the design is illustrated in the setting of severe arthritis of the hip trials.


Salmonella serotypes in the environment.Cats can shed antimicrobial drug−resistant  Salmonella to humans, we evaluated the excretion of Salmonella by pet cats. Rectal-swab specimens were taken from 278 healthy house cats, from 58 cats that died of disease, and from 35 group-housed cats. Group-housed cats were kept in one room with three cat trays and a common water and feed tray. Eighteen (51.4%) of 35 group-housed cats, 5 (8.6%) of 58 diseased cats (5/58), and 1 (0.36%) of 278 healthy house cats excreted Salmonella. Salmonella isolates were of serotypes Typhimurium, Enteritidis, Bovismorbificans and 4:i:-. Acquired antimicrobial resistance was found in serotype Typhimurium  and 4:i:- strains . Cats that excrete Salmonella can pose a public health hazard to people who are highly susceptible to Salmonella, such as children, the elderly, and immunocompromised persons.To determine whether cats were a risk for transmission of  Salmonella infections are still a leading cause of human foodborne infections in the world , which came from one owner. Finally, rectal samples of 35 kittens  were taken at a facility where the animals were group-housed, waiting to be adopted. These animals came from 16 different owners.Bacteriologic analysis was performed by enrichment of the rectal swabs. The samples were first pre-enriched in buffered peptone water (BPW)  overnight at 37°C, after which 1 mL of this suspension was added to 9 mL of tetrathionate brilliant green broth (Oxoid) (enrichment). After incubation overnight at 37°C, a drop of this suspension was spread on brilliant green agar (BGA) (Oxoid). Both the serotype and phage type of positive isolates were determined.Salmonella enterica serovar Typhimurium 8420 (resistance type ACSSuT), 6237 (sensitive), 3520 (resistance type T), 2200 (resistance type ASSuT), and 5833 (sensitive) isolates from human patients in Belgium were used as control strains in antimicrobial susceptibility testing.Resistance to antimicrobial agents was tested by using the disk diffusion assay on Mueller-Hinton agar with commercial antimicrobial susceptibility disks (Oxoid) according to the international standards of the National Council for Clinical Laboratory Standards (NCCLS) , according to the manufacturer's instructions. All the resistant strains were tested for the presence of the genes typical for particular resistance. The genes determined and primers used are listed in S. Typhimurium F98 was used as a negative control in all the amplifications. S. Typhimurium strains 8420, 6237, 3520, 2200, and 5833 were used as positive controls.For PCR, a loop of bacterial culture was resuspended in 50 μL of water, and DNA was released from bacterial cells by boiling for 20 min. After the mixture was spun for 1 min in a microfuge at 14,000 x Salmonella strains were tested for the presence of the SopB gene. The primers were GATAGGAAAGATTGAGCACCTCTG and TACAGAGCTTCTATCACTCAGCTTC, and the PCR cycle consisted of 30 cycles of .All XbaI PFGE patterns were determined for all 21 S. Typhimurium strains by using previously described PFGE methods . The Salmonella isolates and the human S. Typhimurium isolates 8420, 6237, 3520, 2200, and 5833 to invade human intestinal epithelial cells was determined. Cells of the human colon carcinoma cell line T84 were seeded in 96-well cell culture plates  at a density of 5.105 cells/mL culture medium  and grown for 24 h. Bacteria were grown for 20 h in LB-medium, after which the suspension was diluted 1:50 in fresh LB-medium. After 4 h of incubation at 37°C, suspensions were centrifuged and resuspended in DMEM with 10% fetal calf serum (FCS). The number of colony-forming units (CFU)/mL was determined by plating 10-fold dilutions on BGA. The suspensions were stored overnight at 4°C. The next day, 106 CFU in 200 μL were added to the T84 cell cultures, which were then centrifuged for 10 min at 1,500 rpm to make close contact between the bacteria and the colon cells. The plates were incubated for 1 h at 37°C and 5% CO2. The cells were then rinsed three times with Hanks' Balanced Salt Solution . Cell culture medium with gentamicin (50 μg/mL) was added, and plates were incubated for 1 h at 37°C and 5% CO2. Hereafter, the cells were rinsed three times with PBS and analyzed with 1% Triton X-100  in distilled water. From this lysate, 10-fold dilution series were made. From each dilution, 6 x 20 μL was added to BGA, to determine the number of CFU Salmonella per mL. The assays were performed in triplicate. The percentage of intracellular bacteria, relative to the number of Salmonella bacteria, initially incubated with the cells, was calculated. The previously mentioned human isolates of S. Typhimurium were used for comparison between the cat isolates and human isolates. Statistical analysis was performed by analysis of variance methods using the SPSS 11.0 software.The capacity of all cat Salmonella strain was isolated, an S. Enteritidis phage type 21 strain, sensitive to all tested antimicrobial drugs. Five strains were isolated from cats that died from or were euthanized because of incurable disease. Feline AIDS (caused by feline immunodeficiency virus [FIV]) was diagnosed in three cats, one died due to feline panleukopenia parvovirus infection, and one was poisoned. Three isolates were identified as being ampicillin-resistant S. Typhimurium phage type 193, harboring the TEMbla gene. They had the same pulsed-field gel electrophoresis (XbaI) pattern, indicating that the isolates were of clonal origin , harboring the TEMbla, cat, sul2, tet(A), and dfrA1 antimicrobial drug–resistance genes. Eighteen strains were isolated from the group-housed cats. All of these were S.Typhimurium phage type 120/ad. Fourteen of these strains showed acquired resistance to ampicillin, chloramphenicol and tetracycline and harbored the TEMbla, cat, and tet(A) antimicrobial drug-resistance genes, while four isolates were resistant to chloramphenicol only and only harbored the cat gene  and the human isolate S. Typhimurium strain 2200, the most invasive, yielding a percentage of invasion of 8% to 10%. The multidrug-resistant cat isolate Salmonella 4:i:- (strain 11) was the least invasive strain, having an invasion percentage of about 0.5%. Invasion percentages of the different isolates are shown in All isolates invaded T84 cells, with the cat isolates of Salmonella strains, healthy house cats are generally safe. Earlier reports regarding isolation of pathogens from healthy cats showed low percentages (mostly around 1%) of Salmonella-positive rectal swabs  of S. Typhimurium 120/ad was isolated, indicates spread of the Salmonella strain between the cats or a common source. The age of these animals may also play a role, since all animals in this group were <4 months. Young animals are more susceptible to Salmonella infection. Also immunodeficiency can result in Salmonella excretion. One outbreak of fatal salmonellosis in cats has been reported after mild immunosuppression induced by live panleukopenia virus vaccination  were resistant to ampicillin, chloramphenicol, and tetracycline. Resistance genes were found to be TEMbla (ampicillin), cat (chloramphenicol), and tet(A) (tetracycline). The genes in the class 1 integron of the multidrug-resistant genomic island in ACSSuT type S. Typhimurium, required for the resistances to the above three mentioned antimicrobial drugs, are PSE1bla, floR, and tet(G) , encoded by TEMbla (ampicillin), cat (chloramphenicol), sul2 (sulfonamides), tet(A) (tetracycline), and dfrA1 (trimethoprim). Also the resistance shown by this example had no relationship to the typical S. Typhimurium DT104 multidrug-resistant genomic island.Since the 1990s, concerns have arisen about the emergence and spread of multidrug-resistant Typhimurium strains, especially the multidrug-resistant ACSSuT type, which is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (Salmonella in the environment. Cats that are sick or are receiving medication resulting in immune deficiencies can potentially pose a threat to public health. Young children, the elderly, and immunocompromised persons are at risk because of their high sensitivity for the infection. All persons should follow good hygiene practices when keeping cats as pets.In conclusion, healthy house cats are generally safe with regard to excretion of


Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity. Lactococcus lactis NCDO 2118 is a nondairy strain, a xylose fermenter (a common trait of plant-associated strains), and a GABA producer isolated from frozen peas (Lactic acid bacteria (LAB), in general, acquire energy from the conversion of sugars into lactic acid  and are zen peas , 7.L. lactis NCDO 2118 was sequenced three times, due to assembling complexity. First, the genome was decoded with the SOLiD 5500 platform with mate-paired libraries, generating a total of 5,133,057,360 bp, . The reads were subjected to a Phred 20 quality filter using Quality Assessment software . Then manual curation was performed using Artemis . Genome assembly was performed using Mira 3.9 . Assembly was performed with Mira 4.0.1 and Newbler 2.9  to perfoL. lactis NCDO 2118 consists of a single circular chromosome of 2,554,693 bp, containing 2,386 coding sequences (CDS), which had 52 pseudogenes, 66 tRNA genes, and 6 rRNA operons, with a G+C content of 34.9%. There is one plasmid, pNCDO2118 , with 48 CDS, from which 4 are pseudogenes with a G+C content of 32.33%.The complete genome of Lactococcus lactis NCDO 2118 chromosome and the plasmid were deposited at DDBJ/EMBL/GenBank under the accession numbers CP009054 and CP009055, respectively.The


Primary care randomised controlled trial of a tailored interactive website for the selfmanagement of respiratory infections (Internet Doctor). BMJ Open 2016;6:e009769. doi:10.1136/bmjopen-2015-009769Little P, Stuart B, Andreou P, There is a labeling mistake in the analysis of this paper. Although the mistake alters the estimates very slightly it does not alter the inferences.“Results 3044 participants were recruited. 852 in the intervention group and 920 in the control group reported one or more RTIs, among whom there a modest increase in NHS Direct contacts in the intervention group (intervention 44/1734 (2.5%) versus control 24/1842 (1.3%); multivariate Risk Ratio (RR) 2.53 ). Conversely reduced contact with doctors occurred (283/1734 (16.3%) vs 368/1845 (20.0%); risk ratio 0.71, 0.53 to 0.95, p=0.019). Reduction in contacts occurred despite slightly longer illness duration  and more days of illness rated moderately bad or worse illness . The estimate of slower symptom resolution in the intervention group was attenuated when controlling for whether individuals had used webpages which advocated ibuprofen use . There was no evidence of increased hospitalisations .”The misunderstanding was that the 24 week follow-up (the last data point) was thought to be 20 weeks, and the real 20 week data was then not included. The most complete use of the data is therefore to include both the 20 and 24 weeks data. If the complete 24 weeks of data is used throughout this means the following changes to the tables: In the Abstract, the Results section should read: Table 2 is the characteristics of those who reported at least one illness and that does not change. The main impact is on the numbers in Table The title of table The corrected Tables are shown below:


High performance, cast aluminum automotive wheels are increasingly being incrementally formed via flow forming/metal spinning at elevated temperatures to improve material properties. With a wide array of processing parameters which can affect both the shape attained and resulting material properties, this type of processing is notoriously difficult to commission. A simplified, light-duty version of the process has been designed and implemented for full-size automotive wheels. The apparatus is intended to assist in understanding the deformation mechanisms and the material response to this type of processing. An experimental protocol has been developed to prepare for, and subsequently perform forming trials and is described for as-cast A356 wheel blanks. The thermal profile attained, along with instrumentation details are provided. Similitude with full-scale forming operations which impart significantly more deformation at faster rates is discussed. One of the more challenging metal forming operations currently being practiced in the aerospace and transportation sectors is metal spinning, including derivatives such as shear forming and flow forming1From a processing standpoint, there are a wide range of parameters which can dictate the shape and properties of the manufactured component. Numerous studies have focused on statistical techniques for optimizing various parameters34Cast aluminum alloys are employed in a wide variety of automotive and aerospace applications, with alloy A356 used in automotive wheels. However, this alloy is not suitable for forming at room temperature69Figure 1). This apparatus has been built primarily from a manual, belt-driven capstan lathe with a total output of 22 kW, and a propane torch heating system with a peak output of 82 kW . A mandrel with embedded thermocouples along with a rigid roller assembly  has been installed, which is capable of forming workpieces up to 330 mm in diameter. The mandrel has a manually activated clamping system which is able to account for large changes in workpiece diameter occurring during processing . A battery operated Data Acquisition (DAQ) system containing a miniature wireless computer capable of monitoring the temperature of the mandrel during forming and the blank for characterizing heating has been installed on the quill of the lathe. While other flow forming processes have been synthesized using adapted lathesin situ heating and thermal data acquisition.In order to support development and optimization of flow forming operations for wheel manufacturing, custom forming equipment has been developed at the Department of Materials Engineering at the University of British Columbia  and main working directions and components labelled on a computer-aided design depiction (bottom). Please click here to view a larger version of this figure.Figure 2: Heating system detail. A propane heating system with four discrete burners (top and bottom right) actuated from a central manifold containing a gas control solenoid (top and bottom left). Gas pressure and a discrete flow rate to each of the burners is possible, along with placement along the blank to conform to different geometries. Please click here to view a larger version of this figure.Figure 3:Roller stand assembly detail. The original tool holder on for the lathe has been adapted to hold a roller at arbitrary angles relative to the turning axis of the mandrel via a jam nut assembly. Please click here to view a larger version of this figure.Figure 4:Instrumented mandrel and clamp system overview. The rotary tooling has been designed to bolt directly to the lathe spindle, which is in turn supported by a live center on the tailstock (top and bottom left). Clamp assembly/operation is also depicted (top and bottom right). Please click here to view a larger version of this figure.Acquire as-cast workpieces machined to the mandrel size such that the inner diameter runout is 0.2 mm, while the outer diameter retains as much cast surface as possible. NOTE: If blanks are drawn from full-size wheel castings, machining operations are required to remove all hub and spoke portions, while providing features which can be employed to clamp the workpiece to the mandrel. This includes removal of the in-board flange.Pre-heat a coffin furnace able to receive the entire workpiece to 135 °C, clean the workpiece with degreaser and place in furnace for an hour to prepare for thermal barrier coating application.Rapidly remove the workpiece from the furnace, and place on a coating jig. Using an automotive-type paint sprayer, apply a thin layer of thermal barrier die coating to the inner diameter. NOTE: This coating will provide lubrication and reduce heat transfer to the mandrel during forming operations.Wipe down the mandrel surface with a damp cloth. Ensure that the mandrel has a total rotational runout of < 0.5 mm using a dial gauge indicator along the forming length. Assess this with a live tooling center engaged on the tailstock plate. Using a torque wrench, ensure that all fasteners aside from those on the clamp assemblies are tightened to specified torque values for Grade 12.9 bolts .Start the pre-heating system by first powering the gas supply solenoid, and then igniting the torches with a flint spark lighter. Run the pre-heating system for 10 min to expel any condensate collected in the torches/hoses. Extinguish by deactivating the gas supply solenoid.Remove any loose/oxidized coating layer on the mandrel with dry 600/P1200 grit silicon carbide paper while turning the mandrel at 20 rotations per minute (rpm).Power the on-board data acquisition module, and run the pre-heating system until the thermocouples embedded in the mandrel surface read 200 °C with the live center engaged.Using an automotive-type paint sprayer, lightly coat the mandrel surface with a water-based forging lubricant and allow the rotary tooling to cool to ambient temperature with the live tooling center engaged.Figure 3) with a wrench. Set the approach or attack angle on the roller assembly using a toolmaker's protractor, and tighten both internal and external nuts (M35 - 750 Nm).Loosen the jam nut assembly on the roller stand  by first engaging the M12 shoulder bolt to connect element 2 to the clamp bracket. Inspect for any thermal distortion which will prevent element 2 in Figure 4 from smoothly running against the clamp bracket. Ensure that they move freely, lightly sanding the contact surfaces with dry 320/P400 grit silicon carbide paper. Apply a thin layer of high temperature molybdenum-based lubricant with a cloth as needed.Assemble the 3 clamp assemblies .Move the roller axially and radially (approx. 2-5 mm from workpiece surface) into position for forming, and perform one last clamp tightening  to capture the distribution of temperature across the surface of the blank during this aspect of the process. This response is shown in Figure 5:Typical temperature profile of mandrel and blank. A representative transient thermal response of the blank and mandrel obtained with the heating system. Vertical dashed lines indicate where clamps were tightened during the preheating steps, and the black arrow depicts forming. The last vertical line shows where the heating system was turned off whilst the system cooled.Figure 6: As-cast and formed result. The as-received, as-cast blank surface and geometry having a minimum inner diameter of 330 mm (top) was deformed in two passes to provide the result shown (middle). The as-cast dendritic microstructure (bottom left) is visibly modified by the forming operation and a subsequent T6 heat treatment (bottom right) as observed with optical microscopy8Please click here to view a larger version of this figure.The representative results shown above highlight that the protocol and equipment employed is capable of forming cast aluminum at elevated temperatures, and has provided a platform to determine a processing window for flow forming of wheels. The technique demonstrated can be used to explore aspects of forming envelopes, including how both formed and unformed material responds to heat treatmentRegarding further instrumentation, which would accelerate process model development, the inclusion of machine-tool dynamometer and tribometers1113As it is largely a manual forming process for a material which is sensitive to time at temperature, some inconsistencies between run to run are to be expected. Aluminum alloys have microstructures that are highly sensitive to temperatures above 100°C due to ageing mechanisms. Therefore, the most critical steps within the protocol are 1.2 and 3.3-3.7, where the blank is at elevated temperatures. Tightening and re-seating the clamps must be conducted as quickly as possible to maintain repeatability between forming operations.in situ workpiece heating employed during the pre-heating step is quite inefficient and could be improved via radiative heating. The overall processing speeds in terms of mandrel and tool movements that can be attained are somewhat limited by the capabilities of the lathe employed. Higher forming speeds require a more rigid frame with a higher load capacity, particularly if the forming of a stronger material were to be attempted. Workpiece clamping and release could be improved with the addition of hydraulic or pneumatic actuation. As heat transfer from the blank to the mandrel is largely a function of the pressure imposed by the workpiece onto the mandrel, this addition could also improve a model-based approach to ascertain the workpiece temperature during forming with the existing system.The The apparatus and procedure described has shown that forming loads for this material under these conditions approaches those for standard turning operations, and remains a very cost effective process by which to perform manufacturing trials. Research into different manufacturing routes and formability can be performed away from commercial forming equipment, which is exceedingly expensive to operate. With the apparatus and protocol described, processing parameters can be investigated prior to constructing larger scale, higher throughput equipment, and to the authors' knowledge, is a unique approach.As the protocol developed has only been applied to one specific variant of cast aluminum alloy, there is a multitude of other aluminum foundry alloys which could be investigated for a variety of applications beyond automotive wheels. As these alloys have approximately similar processing windows from a temperature perspective, the protocol developed can be readily adapted.The authors have nothing to disclose.


The National Institute for Health Research (NIHR) has identified a gap in the number of people it funds who are on a pathway to become future leaders of clinical trials, compared to how much the NIHR invests in clinical trials. In order to support the clinical trials of tomorrow, it is vital that the right people are supported now to lead these trials. To address this issue, NIHR organised a workshop with key stakeholders to understand the barriers to embarking on a clinical trials career and explore initiatives to increase capacity and capability in clinical trials. The output from the workshop was a set of recommendations which NIHR is now considering to shape future support. The vision of the NIHR is, ‘To improve the health and wealth of the nation through research’, and one of its aims in order to realise this vision is to ‘Attract, develop and retain the best research professionals to conduct people-based research’. One of the main ways in which NIHR does this is through its research training programmes which are designed to create the applied health research leaders of the future (www.nihr.ac.uk/funding/training-programmes.htm). The NIHR does not provide training  through these programmes; rather, it provides funding and support to allow the best people to undertake excellent research and extensive training and development programmes based within high-calibre organisations. There are funding opportunities available for all professions, clinical and non-clinical, from masters to professorial level. These opportunities allow people to undertake research training through doing research across a range of disciplines and research methodologies. Training in clinical trials represents an important area in which people undertake training as part of an NIHR research training award. Throughout this letter where training in clinical trials is mentioned, this refers to the training undertaken as part of a NIHR research training award, not training provided directly by NIHR. Details of the guidance currently given to applicants for NIHR research training awards can be found within the latest application guidance notes (e.g. http://www.nihr.ac.uk/documents/funding/Training-Programmes/TCC-Fellowships-Guidance-Notes-2016.pdf).The National Institute for Health Research (NIHR) is the research arm of the National Health Service (NHS) in England. It is funded by the Department of Health in order to deliver the Government’s strategy for applied health research  where appropriate and must have the right level of trials experience in their supervisory team. Applicants are also encouraged to think about the scope of any trial or feasibility study in relation to their research training award to ensure that it is realistic to complete within the award timescale and whether it represents a good training vehicle. However, analysis of the NIHR training portfolio, by looking at the type of research proposed within research training award applications, has identified a gap in the number of people undertaking trials training, with approximately 20 % of funded fellowships including a trial or feasibility study. This compares to 59 % of completed Research for Patient Benefit (RfPB) grants being feasibility studies or randomised controlled trials (RCTs) as of October 2013. The RfPB programme represents a first opportunity for many NIHR trainees to gain experience of applying for and being awarded a significant research grant, and whilst it is not necessarily expected that the profile of research training awards in terms of research areas  and methodologies  should exactly mirror that of the NIHR research programmes, it is NIHR’s view that they should broadly align. This will ensure that the profile of new researchers who will become the research leaders of tomorrow are able to match the requirements, in terms of skills and expertise, needed to deliver future NIHR research. Considering the amount of investment by NIHR in RCTs (774 active RCTs in May 2014 with a research cost of £880 million), it is very important that NIHR trains the individuals capable of leading trials in the future. To help understand barriers to individuals undertaking trials training and to explore initiatives to increase capacity and capability in clinical trials amongst trainees, NIHR set up a workshop with key stakeholders which was held on 29 June 2015. Details of the participants are given in Table Share the current provision for trainees interested in a career as a clinical trialistUnderstand any barriers to trainees embarking on a career in clinical trialsUnderstand any barriers to institutions supporting trainees involved in clinical trialsExplore initiatives to increase capacity and capability in clinical trials amongst NIHR traineesThe aims of the workshop were to:The workshop heard views from across NIHR about the current provision for clinical trials training and potential ways forward, including the current provision of research training awards and the viewpoints of a CTU director, a current NIHR trainee, from NIHR’s research programmes and the NIHR Clinical Research Network. The discussion then focussed on how a trial fits into a fellowship and whether this represents a good training vehicle for someone interested in becoming a future trials leader. Training awards which do include a clinical trial primarily focus on feasibility studies and less on full trials. A lot of discussion took place focussed on what a fellowship based around clinical trials training should look like; for example, some successful applicants have used a fellowship to undertake a lot of the groundwork and preparatory research before going onto to gain further funding for a feasibility, pilot study or full trial. The benefit of working with a CTU to gain experience in a wide range of trial activities was also discussed.Are training programmes fit for purpose?What does a career pathway look like?How do trainees/researchers move through the NIHR pipeline?The second part of the workshop was structured into breakout groups to discuss the following points:Each of the breakout groups had a facilitator who recorded the key points from each discussion. Each group nominated a spokesperson to feed back to the wider group the key points recorded, which informed a further discussion by the whole group. The meeting Chair facilitated these discussions, and recommendations were agreed by consensus decision-making.In looking at the training programmes, the workshop concluded that work should be done to investigate providing more flexibility for people wanting to apply for a NIHR Clinical Trials Fellowship (CTF). NIHR CTFs are 6-month fellowships open to already funded NIHR trainees who are interested in undertaking an intense period of clinical trials training in partnership with a CTU following the conclusion of their current NIHR research training award. The workshop also concluded that consideration should be given to expanding the scope of the NIHR Transitional Research Fellowship (TRF) to provide a route into clinical trials for post-doctoral trainees with little trials experience. NIHR TRFs are currently targeted at researchers from a basic science background who want to transition into applied health research and at researchers returning from a significant career break. Undertaking a TRF with focus on moving into clinical trials could then put applicants in a strong position to apply for further trials funding or fellowships, to lead on a small feasibility study, for example. To ensure that training takes place within a high-quality environment, encouragement, or a requirement, to link with a CTU could be given. The possibility of providing fellowships with explicit links to already funded trials was also discussed.In thinking about career pathways, the workshop considered only those training to become future Chief Investigators rather than the myriad of other roles involved with clinical trials. It was agreed that a clear career pathway for future Chief Investigators does not currently exist and that career pathways for researchers who undertake trials training is currently based more upon their clinical academic pathway (for clinician researchers). Expanding the scope of the TRF as described above could help with this, as would wider dissemination of case studies showcasing people who have forged out a career in clinical trials.Discussions around moving trainees through the NIHR pathway focussed again on those training to become future Chief Investigators. There was an agreement that training awards should have a focus on feasibility and the wider aspects of clinical trials so that trainees are in a strong position to apply for trials funding in the future from programmes like Health Technology Assessment (HTA). The group also thought that developing some key skills for those training in clinical trials would be helpful. This could help, for example, someone wanting to put together a PhD programme with a focus on trials training.The outcome of the workshop was a set of recommendations that have been developed into a specific set of proposals through a task and finish group which NIHR set up following the workshop. These proposals are now under consideration by NIHR, and any changes to research training programmes as a result of these proposals will be announced during course of 2016.Share the current provision for trainees interested in a career as a clinical trialistUnderstand any barriers to trainees embarking on a career in clinical trialsUnderstand any barriers to institutions supporting trainees involved in clinical trialsExplore initiatives to increase capacity and capability in clinical trials amongst NIHR traineesThe original aims of the workshop had been to:The timescale of a clinical trial does not necessarily fit with that for a personal research training award.The additional staff and funds required to run a large trial do not necessarily fit within the scope of what can be provided by a personal research training award.The additional time constraints associated with registering for a PhD are not always compatible with the timescales of a clinical trial.Undertaking a clinical trial or research on a trials-related topic may not be as attractive to potential PhD students as more traditional lab-based research.Applicants are not necessarily aware of how current NIHR awards can be utilised to further skills and experience in clinical trials.There is not a clear career pathway for researchers looking to become future Chief Investigators.Additional flexibility may be required in some schemes, particularly the CTF, to increase their attractiveness to potential applicants.The workshop represented a good opportunity to present the current provision of research training opportunities provided by NIHR which allow for training in clinical trials, and as a result of the discussions, several potential barriers to people taking up these opportunities emerged. These are summarised below:The identification of potential barriers helped shape the recommendations which came out of the workshop, all of which were agreed upon with the aim of overcoming these barriers and increasing capacity and capability in clinical trials amongst NIHR trainees.Increase flexibility and availability of training programmes for clinical trials trainingIncrease dissemination of opportunities for clinical trials trainingExplore linking trials training into the broader research pathwayConsider key skills for different stages of clinical trials trainingConsider how research training awards can best prepare trainees for a career as a trials leader when developing recommendations into concrete proposalsThese recommendations are broadly summarised below:To develop proposals based upon the recommendations of the NIHR Clinical Trials Training workshop for future clinical trials training within NIHRTo advise on the implementation of these proposals within the current structure of NIHR research training programmesThe task and finish group which developed these recommendations further was made up of a sub-section of the attendees at the workshop reported here and worked to the following terms of reference:Whilst there are no plans to repeat the workshop reported here, any changes that are implemented as a result of this workshop will be reviewed in the future and where appropriate, and if required, further workshops may be organised.CTF, Clinical Trials Fellowship; CTU, Clinical Trials Unit; HTA, Health Technology Assessment; NHS, National Health Service; NIHR, National Institute for Health Research; RCT, randomised controlled trial; RfPB, Research for Patient Benefit; TRF, Transitional Research Fellowship


Clostridium perfringens enterotoxin (CPE) can be used to eliminate carcinoma cells that overexpress on their cell surface CPE receptors – a subset of claudins . However, CPE cannot target tumors expressing solely CPE‐insensitive claudins (such as Cldn1 and Cldn5). To overcome this limitation, structure‐guided modifications were used to generate CPE variants that can strongly bind to Cldn1, Cldn2 and/or Cldn5, while maintaining the ability to bind Cldn3 and Cldn4. This enabled (a) targeting of the most frequent endocrine malignancy, namely, Cldn1‐overexpressing thyroid cancer, and (b) improved targeting of the most common cancer type worldwide, non‐small‐cell lung cancer (NSCLC), which is characterized by high expression of several claudins, including Cldn1 and Cldn5. Different CPE variants, including the novel mutant CPE‐Mut3 (S231R/S313H), were applied on thyroid cancer (K1 cells) and NSCLC (PC‐9 cells) models. In vitro, CPE‐Mut3, but not CPEwt, showed Cldn1‐dependent binding and cytotoxicity toward K1 cells. For PC‐9 cells, CPE‐Mut3 improved claudin‐dependent cytotoxic targeting, when compared to CPEwt. In vivo, intratumoral injection of CPE‐Mut3 in xenograft models bearing K1 or PC‐9 tumors induced necrosis and reduced the growth of both tumor types. Thus, directed modification of CPE enables eradication of tumor entities that cannot be targeted by CPEwt, for instance, Cldn1‐overexpressing thyroid cancer by using the novel CPE‐Mut3. Clostridium perfringens enterotoxin (CPE) is used to target carcinomas overexpressing a claudin subset serving as CPE receptors. CPE‐based pores in membrane cause cell death. Structure‐guided CPE modifications (CPE‐S231R/S313H) enabled also claudin‐1 binding and growth reduction of claudin‐1‐expressing papillary thyroid carcinoma (mouse xenotransplants) that could not be targeted by CPEwt. Furthermore, CPE‐S231R/S313H improved targeting of lung cancer (NSCLC) expressing multiple claudins. Papillary thyroid carcinoma (PTC), a differentiated type of thyroid cancer, is the most common histological subtype accounting for approximately 70% of thyroid cancer cases  is the most common cancer  and is the most common cause of death from cancer worldwide. In addition, approximately 50–70% of patients with lung adenocarcinoma, one type of NSCLC, after surgery relapse within one year and their cancer cells acquire a chemoresistant phenotype   and two functional domains with a known structure  to Cldn3, Cldn4, Cldn6, Cldn7, and Cldn9; with medium affinity to Cldn8, Cldn14, and Cldn19; and with low affinity to Cldn1 and Cldn2, but does not bind to Cldn5, Cldn10 to Cldn13, Cldn15 to Cldn18, and Cldn20 to Cldn24  and often deregulated Kwon, , therebyet al., et al., et al., et al., et al., In case of thyroid cancer  for cancer treatment is restricted to carcinomas, which express CPEwt‐binding claudins . In addition, extrajunctional CPE‐sensitive claudin molecules are also present on normal epithelia, at least to a minor extent. This can result in side effects during CPE treatment. To overcome these difficulties, we modified CPE for the first time to adjust its claudin‐binding properties to the claudin expression profile of certain cancer types containing claudin subtypes that are not bound by CPEwt.In vitro and in vivo, tumor growth of PTC (K1 cells), overexpressing mainly Cldn1, but not Cldn3 and Cldn4, was efficiently reduced by Cldn1‐binding CPE‐variant S231R/S313H (CPE‐Mut3), but not by CPEwt. In addition, we show that CPE variants suppress growth of NSCLC tumors in a claudin‐directed manner.Here, we demonstrate expansion of CPE‐based oncoleaking strategy specifically for tumor types overexpressing claudins, which are not CPE receptors such as Cldn1 by design and use of CPE mutants: 22.1et al., et al., Plasmid encoding pGEX‐4T‐CPE194‐319 carrying cCPE with N‐terminal GST‐fusions was described  and purified from lysates using Ni‐NTA‐Agarose , as described earlier . Native HEK293 (HEK) cells and HEK cells, stably transfected with Cldn1‐7, were cultured as described elsewhere  precoated 96‐well plates reached 80% confluency, they were transiently transfected with 0.1 µg DNA of CPE constructs and vector with antisense CPE sequence (as negative control) using ViaFect .When cells seeded on poly‐2.5l‐lysine. On next day, cells were preincubated at 37 °C, 5% CO2 for 1 or 24 h, with variable dilutions of cCPE or CPE preparations or 0.01% (v/v) Triton X‐100 (background correction) before medium was replaced by medium without phenol red containing 1.25 mm MTT. Two hours later, cells were treated with 5% (v/v) Triton X‐100 in 2‐propanol for 20 min and absorption was measured at 560 nm.For MTT ‐2,5‐diphenyltetrazolium bromide; Sigma‐Aldrich, USA) cytotoxicity assay cells were seeded 80% confluent on 96‐well plates  precoated with poly‐2.6et al., −1 GST‐cCPE constructs  in 24‐well plates . Amount of bound cCPE was detected as described elsewhere . After reaching confluency, cells were washed and harvested, and extracted membrane protein fraction was analyzed as described elsewhere . Tumor volume  and body weight were determined at least twice weekly. When tumors reached mean volume of 0.14–0.17 cm3, recombinant CPE was injected intratumorally in anesthetized animals. In total, 20 μg of recombinant CPE was applied by 10 injections (daily dose of 0.04 μg CPE·μL−1 0.9% NaCl). During treatment, TV was measured at days 11, 14, 18, and 21 after injection of PC‐9 cells or at days 18, 21, 25, and 28 after injection of K1 cells. Animals were sacrificed for tumor removal and further analysis. All experiments were performed at EPO GmbH, Berlin, Germany.For cell line‐derived subcutaneous xenotransplant (CDX) tumor models, 5 × 102.10Shock frozen tumor tissues were fixed with Tissue‐Tek Medium  and dissected with cryostat into cryosections each of 10 or 18 μm thickness, which were transferred onto cover slides.2.11et al., qupath software  in triplicates of two samples per group. Necrotic area in % related to the whole tissue area is shown.For morphological evaluation of 18 µm thick tissue sections, hematoxylin and eosin (HE) staining was performed. Staining procedures were performed by standard protocols  using one‐way or two‐way ANOVA and Bonferroni’s adjustment for multiple comparisons.Data are expressed as mean ± SEM. Statistical analyses were performed with 33.1et al., et al., We first aimed to create variants of the nontoxic claudin‐binding domain of CPE (cCPE) with increased Cldn1 binding. Mutation S313H in cCPE, was previously shown to improve Cldn1 binding and enable Cldn5 binding (Protze To further improve Cldn1 and Cldn5 binding of cCPE‐Mut1 (cCPE‐S313H), we generated models of claudin–cCPE‐S313H interaction , previously shown to increase binding of cCPE to Cldn1 and other claudins, similar as S313H  and Nthy‐ori 3‐1 cells , PC‐9 (lung adenocarcinoma), and SK‐Mes‐1 (lung squamous‐cell carcinoma). Expression of Cldn1, as well as of other claudins (Cldn2 to Cldn9), in these cell lines was analyzed by western blot Fig. A. For K1Since Cldn1 expression in PC‐9 cells was higher than that of SK‐Mes‐1 cells, PC‐9 cells were used as model for NSCLC for further experiments. K1 cells were chosen as model for PTC and Nthy‐ori 3‐1 cells as thyroid follicular epithelial cell model.3.4First, we tested binding of nontoxic cCPE variants to K1 and Nthy‐ori 3‐1 cells expressing Cldn1. Similarly as found for HEK‐Cldn1 cells Fig. A, cCPE‐MConsequently, cytotoxicity of full length CPE‐Mut3 was tested on Nthy‐ori 3‐1 and K1 cells using MTT assays. After 24‐h incubation with CPEwt, CPE‐negative control, CPE‐Mut3, and CPE‐Mut4, viability of Nthy‐ori 3‐1 cells was not affected Fig. A. In conTo further verify that CPE‐mediated cytotoxicity depends on presence of claudins on surface of K1 cells, K1 cells were incubated simultaneously with both noncytotoxic cCPE (wt or Mut3) and cytotoxic full length CPE‐Mut3 in same molar concentrations. cCPE‐Mut3 bound to Cldn1 on the surface of K1 cells and thereby protected them completely from cytotoxic effect of full length CPE‐Mut3 Fig. B, blue. in vitro PTC model). The insensitivity of Nthy‐ori 3‐1 cells to CPE‐Mut3 is likely due to their low Cldn1 expression and suggests that CPE‐Mut3‐mediated cytotoxicity could potentially be restricted to Cldn1‐overexpressing malignant PTC.Thus, unlike CPEwt and the broad specificity binder CPE‐Mut4, the designed CPE‐Mut3 strongly reduced cell viability selectively in K1 cells overexpressing Cldn1  demonstrated for PC‐9 cells (a) claudin‐dependent cytotoxicity of CPE and (b) that all claudin‐sensitive CPE variants  reduced cell viability. The latter can be explained by the fact that PC‐9 cells do not only express Cldn1, but also Cldn3, Cldn4, and Cldn7  or of vector control preparations were administrated within 10 days (single 2 µg injection daily).Finally, we tested 3 and to 1.0 ± 0.21 cm3, respectively. In contrast, for CPE‐Mut3‐treated animals, only minor tumor growth (to 0.28 ± 0.04 cm3) was observed. The study revealed no significant effect of CPEwt treatment, but a very efficient anti‐tumoral activity of CPE‐Mut3, reflected by reduced tumor growth (14% compared to control)  Fig. A. Hence,In the PC‐9 xenograft model, measurements of TV on days 11, 14, 18, and 21 after beginning of CPE‐administration in different treatment groups revealed anti‐tumoral activity of both CPEwt and of CPE‐Mut3, reflected by respective TVs of 43% and 29%  to expand use of CPE to targeting of Cldn1‐expressing thyroid tumors and (b) to potentially reduce CPE side effects by matching the binding properties of CPE to the claudin expression profile of particular carcinoma types.In this study, we used structure‐guided mutagenesis of CPE to enable for the first time cytotoxic targeting of tumors expressing Cldn1 or Cldn5 to which CPEwt does not bind considerably. As a proof of principle, we showed et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., This study aimed to overcome the restriction of the promising CPE treatment to tumors expressing only receptors for CPEwt  and of NSCLC (PC‐9 cells). For the first time, CPE was used to successfully reduce growth of lung tumors (PC‐9 cells) and its Mut3 enabled efficient Cldn1‐directed reduction of PTC (K1 cells) growth.Furthermore, in contrast to CPEwt, CPE‐Mut3 was cytotoxic for K1 thyroid cancer cells expressing endogenous Cldn1. For PC‐9 lung cancer cells that express multiple claudins, apart from CPE‐Mut3 also CPEwt and CPE‐Mut4 mediated cytotoxicity. Finally, claudin‐specific cytotoxicity was confirmed Using the same approach of structure‐guided mutagenesis, we also generated CPE‐Mut9 and CPE‐Mut10 which enabled efficient Cldn5‐ and Cldn4‐directed cytotoxicity, respectively Fig. . Thus, Cet al., et al., et al., et al., In several studies, high claudin specificity of anti‐claudin antibodies  as the recombinant protein  by subsequent use of cytotoxic cCPE‐PSIF (protein synthesis inhibitory factor) fusion proteins .All animal experimental procedures are performed in accordance with the in‐house guidelines of the Institutional Animal Care and in accordance with the German Animal Protection Law and approved by the local responsible and official authorities, State Office of Health and Social Affairs .Fig. S1. Shift of claudin‐subtype‐directed cytotoxicity of CPE by structure‐guided mutation of CPE.Fig. S2. Claudin‐subtype‐directed cytotoxicity of CPE variants is mediated by induction of Ca2+ influx.Fig. S3. Visualization of CPE and claudins in thyroid and lung xenograft models after recombinant CPE treatment.Table S1. cCPE or CPE variants and claudin‐binding characteristics.Click here for additional data file.