Survivin

Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, is a protein that, in humans, is encoded by the BIRC5 gene. NCBI Reference Sequence: NG_029069.1

Survivin is a member of the inhibitor of apoptosis (IAP) family. The survivin protein functions to inhibit caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death. This has been shown by disruption of survivin induction pathways leading to increase in apoptosis and decrease in tumour growth. The survivin protein is expressed highly in most human tumours and fetal tissue, but is completely absent in terminally differentiated cells. These data suggest survivin might provide a new target for cancer therapy that would discriminate between transformed and normal cells. Survivin expression is also highly regulated by the cell cycle and is only expressed in the G2-M phase. It is known that Survivin localizes to the mitotic spindle by interaction with tubulin during mitosis and may play a contributing role in regulating mitosis. The molecular mechanisms of survivin regulation are still not well understood, but regulation of survivin seems to be linked to the p53 protein. It also is a direct target gene of the Wnt pathway and is upregulated by beta-catenin.

IAP family of anti-apoptotic proteins

Survivin is a member of the IAP family of antiapoptotic proteins. It is shown to be conserved in function across evolution as homologues of the protein are found both in vertebrates and invertebrates. The first members of the IAPs identified were from the baculovirus IAPs, Cp-IAP and Op-IAP, which bind to and inhibit caspases as a mechanism that contributes to its efficient infection and replication cycle in the host. Later, five more human IAPs that included XIAP, c-IAPl, C-IAP2, NAIP, and survivin were discovered. Survivin, like the others, was discovered by its structural homology to IAP family of proteins in human B-cell lymphoma. The human IAPs, XIAP, c-IAPl, C-IAP2 have been shown to bind to caspase-3 and -7, which are the effector caspases in the signaling pathway of apoptosis. It is not known with absolute certainty though, how the IAPs inhibit apoptosis mechanistically at the molecular level.

A common feature that is present in all IAPs in the presence of a BIR (Baculovirus IAP Repeat, a ~70 amino acid motif) in one to three copies. It was shown by Tamm et al. that knocking out BIR2 from XIAP was enough to cause a loss of function in terms of XIAPs ability to inhibit caspases. This gives the implication that it is within these BIR motifs that contains the anti-apoptotic function of these IAPs. Survivin's one BIR domain shows a similar sequence compared to that of XIAP's BIR domains.

Isoforms

The single survivin gene can give rise to four different alternatively spliced transcripts:

 Survivin, which has a three-intron–four-exon structure in both the mouse and human.
 Survivin-2B, which has an insertion of an alternative exon 2.
 Survivin-Delta-Ex-3, which has exon 3 removed. The removal of exon 3 results in a frame shift that generates a unique carboxyl terminus with a new function. This new function may involve a nuclear localization signal. Moreover, a mitochondrial localization signal is also generated.
 Survivin-3B, which has an insertion of an alternative exon 3.

Structure

A structural feature common to all IAP family proteins is that they all contain at least one baculoviral IAP repeat (BIR) domain characterized by a conserved zinc-coordinating Cys/His motif at the N-terminal half of the protein.

Survivin is distinguished from other IAP family members in that it has only one BIR domain. The mice and human BIR domain of survivin are very similar structurally except for two differences that may affect function variability. The human survivin also contains an elongated C-terminal helix comprising 42 amino acids. Survivin is 16.5 kDa large and is the smallest member of the IAP family.
X-ray crystallography has shown two molecules of human surviving coming together to form a bowtie-shape dimer through a hydrophobic interface. This interface includes N-terminal residues 6-10 just before the BIR domain region and the 10 residue region connecting the BIR domain to the C-terminal helix. The structural integrity of the determined crystal structure of survivin is quite reliable, as physiological conditions were used to obtain the images.

Function

Apoptosis

Apoptosis, the process of programmed cell death, involves complex signaling pathways and cascades of molecular events. This process is needed for proper development during embryonic and fetal growth where there is destruction and reconstruction of cellular structures. In adult organisms, apoptosis is needed to maintain differentiated tissue by striking the balance between proliferation and cell death. It is known that intracellular proteases called caspases degrade the cellular contents of the cell by proteolysis upon activation of the death pathway.

Mammalian cells have two main pathways that lead to apoptosis.

1. Extrinsic Pathway: Initiated by extrinsic ligands binding to death receptors on the surface of the cell. An example of this is the binding of tumour necrosis factor-alpha (TNF-alpha) to TNF-alpha receptor. An example of a TNF receptor is Fas (CD95), which recruits activator caspases like caspase-8 upon binding TNF at the cell surface. The activation of the initiator caspases then initiates a downstream cascade of events that results in the induction of effector caspases that function in apoptosis.

2. Intrinsic Pathway: This pathway is initiated by intracellular or environmental stimuli. It is focused on detecting the improper functioning of the mitochondria in the cell and, as a result, activates signaling pathways to commit suicide. The membrane permeability of the mitochondria increases and particular proteins are released into the cytoplasm that facilitates the activation of initiator caspases. The particular protein released from the mitochondria is cytochrome c. Cytochrome c then binds to Apaf-1 in the cytosol and results in the activation of initiator caspase-9. The activation of the initiator caspases then initiates a downstream cascade of events that results in the induction of effector caspases that function in apoptosis.

One family of proteins called IAPs plays a role in regulating cell death by inhibiting the process. IAPs like survivin, inhibit apoptosis by physically binding to and inhibiting proper caspase function. The function of IAPs is evolutionarily conserved as Drosophila homologues of IAPs have been shown to be essential for cell survival.

IAPs have been implicated in studies to have a regulatory effect on cell division. Yeast cells with knock-outs of certain IAP genes did not show problems associated with cell death, but showed defects in mitosis characterized by improper chromosome segregation or failed cytokinesis.

Deletion of particular IAPs does not seem to have a profound effect on the cell-death pathway as there is a redundancy of function by the many IAPs that exist in a cell. They have been implicated, however, to play a role in maintaining an anti-apoptotic environment intracellularly. Changing the expression of particular IAPs has shown an increase in spontaneous cell death induction or increased sensitivity to death stimuli.

Mechanism of action

Inhibition of Bax and Fas-induced apoptosis

Tamm et al. have shown that survivin inhibits both Bax and Fas-induced apoptotic pathways. The experiment involved transfecting HEK 293 cells with a Bax-encoding plasmid, which resulted in an increase in apoptosis (~7 fold) as measured by DAPI staining. They then contransfected the 293 cells with Bax-encoding plasmid and survivin-encoding plasmids. They observed that cells transfected along with the survivin showed a significant decrease in apoptosis (~3 fold). A similar result also showed for cells transfected with the Fas-overexpressing plasmid. Immunoblots were performed and confirmed that survivin does not inhibit by mechanism of preventing Bax or Fas protein from being made into fully functional proteins. Therefore, survivin should be acting somewhere downstream of the Bax or Fas signaling pathway to inhibit apoptosis through these pathways.

Interaction with caspase-3 and -7

In this part of the experiment, Tamm et al. transfected 293 cells with survivin and lysed them to obtain cell lysate. The lysates were incubated with different caspase forms and survivin was immunopercipitated with anti-survivin antibody. The idea behind this is that, if survivin binds physically with the caspase it is incubated with, it will be co-precipitated along with the survivin while everything else in the lysate is washed away. The immunoprecipitates were then run on SDS-PAGE and then immunoblotted for detection of the desired caspase. If the caspase of interest was detected, it meant that it was bound to survivin in the immunoprecipitation step implicating that survivin and the particular caspase had bound beforehand. Active caspase-3 and -7 coimmunoprecipitated with survivin. The inactive proforms of caspase-3 and -7 did not bind survivin. Survivin also does not bind to active caspase-8. Caspase-3 and -7 are effector proteases whereas caspase-8 is an initiator caspase that sits more upstream in the apoptotic pathway. These results demonstrate survivin's capability to bind with particular caspases in vitro, but may not necessarily translate over to actual physiological conditions. Later, a 2001 study confirmed that human survivin tightly binds caspase-3 and -7 when expressed in E. coli.

Further evidence to support the idea that survivin blocks apoptosis by directly inhibiting caspases was given by Tamm et al. 293 cells were transfected with either overexposed caspase-3 or -7 encoding plasmid and with survivin. They showed that survivin inhibited processing of these two caspases into their active forms. While survivin has been shown as mentioned above to bind to only the active forms of these caspases, it is likely here that survivin inhibits the active forms of the caspases resulting from cleaving and activating more of its own proforms. Thus, survivin acts possibly by preventing such a cascade of cleavage and activation amplification from happening resulting in decreased apoptosis.

In similar manner, looking at the mitochondrial pathway of apoptosis, cytochrome c was transiently expressed in 293 cells to look at the inhibitory effects survivin had on this pathway. Although the details are not here, survivin was shown to also inhibit cytochrome c and caspase-8-induced activation of caspases.

Regulation of cytokinesis

While the mechanism by which survivin may regulate cell mitosis and cytokinesis is not known, the observations made on its localization during mitosis suggests strongly that it is involved in some way in the cytokinetic process.

Proliferating Daoy cells were placed on a glass coverslip, fixed and stained with fluorescent antibodies for survivin and alpha-tubulin. Immunoflourescence using confocal microscopy was used to look at the localization of survivin and tubulin during the cell-cycle to look for any patterns of survivin expression. Survivin was absent in interphase, but present in the G2-M phase.

During the different stages of mitosis, one could see that survivin follows a certain localization pattern. At prophase and metaphase, survivin is mainly nuclear in location. During prophase, as the chromatin condenses so that it is visible under the microscope, survivin starts to move to the centromeres. At prometaphase when the nuclear membrane dissociates and spindle microtubules cross over the nuclear region, survivin stays put at the centromeres. At metaphase, when the chromosomes align at the middle plate and are pulled with high tension to either pole by the kinetochore attachments, survivin then associates with the kinetochores. At anaphase as separation of the chromatids happens, the kinetochore microtubules shorten as the chromosomes move towards to the spindle poles and survivin also moves along to the midplate. Survivin thus accumulates at the midplate at telophase. Finally, survivin localizes to the midbody at the cleavage furrow.

Interaction and localization to the mitochondria

It has been shown that survivin can heterodimerize individually with the two splice variants Survivin-2B and survivin-deltaEx3. Evidence of the heterodimerization of survivin splice variants with survivin was shown with co-immunoprecipitation experiments after cotransfection with the respective survivin variants with survivin. To determine the localization of exogenously expressed survivin-2B and survivin-deltaEx3, fusion constructs of the proteins were made with GFP and HcRed respectively and Daoy cells were transfected with the plasmid constructs. Survivin was also tagged with a fluorescent protein. The fusion of the survivin variants with the fluorescent molecules allows for simple detection of cellular location by fluorescence microscopy. Survivin-2B by itself, localized to both nuclear and cytoplasmic compartments whereas survivin-deltaEx3 localized only in the nucleus. The localization of the three variants (survivin, Survivin-2B, and survivin-deltaEx3) differ, however, when cotransfected together rather than individually.

To see which subcellular compartments contained the survivin splice variants complexes, fluorescent antibody markers for different organelles in the cell were employed. The assumption is that, under fluorescence microscopy, if the particular survivin complex is located in that particular cell compartment, one would observe an overlap from the fluorescence given off by the tagged survivin complex and the tagged compartment as well. Different color fluorescence is used to distinguish compartment from survivin.

 Endoplasmic reticulum and lyosomes: no colocalization
 Mitochondria and golgi: both survivin/survivin-2B and survivin/survivin-deltaEx3 colocalize

To verify these observations, they fractionated the subcellular compartments and performed western blot analysis to definitively say that survivin complexes did indeed localize at these compartments.

Role in cancer

Expression in different carcinomas

Survivin is known to be expressed during fetal development and across most tumour cell types, but is rarely present in normal, non-malignant adult cells. Tamm et al. showed that survivin was expressed in all 60 different human tumour lines used in the National Cancer Institute's cancer drug-screening program, with the highest levels of expression in breast and lung cancer lines and the lowest levels in renal cancers. Knowing the relative expression levels of survivin in different tumour types may prove helpful as survivin-related therapy may be administered depending on the expression level and reliance of the tumour type on survivin for resistance to apoptosis.

As an oncogene

Survivin can be regarded as an oncogene as its aberrant overexpression in most cancer cells contributes to their resistance to apoptotic stimuli and chemotherapeutic therapies, thus contributing to their ongoing survival.

Genomic instability

Most human cancers have been found to have gains and losses of chromosomes that may be due to chromosomal instability (CIN). One of the things that cause CIN is the inactivation of genes that control the proper segregation of the sister chromatids during mitosis. In gaining a better understanding of survivin's function in mitotic regulation, scientists have looked into the area of genomic instability. It is known that survivin associates with microtubules of the mitotic spindle at the start of mitosis.

It has been shown in the literature that knocking out survivin in cancer cells will disrupt microtubule formation and result in polyploidy as well as massive apoptosis. It has also been shown that survivin-depleted cells exit mitosis without achieving proper chromosome alignment and then reforms single tetraploid nuclei. Further evidence also suggests that survivin is needed for sustaining mitotic arrest upon encounter with mitosis problems. The evidence mentioned above implicates that survivin plays an important regulatory role both in the progression of mitosis and sustaining mitotic arrest. This seems strange, as survivin is known to be highly upregulated in most cancer cells (that usually contain chromosome instability characteristics), and its function is that which promotes proper regulation of mitosis.

Regulation by p53

p53 inhibits survivin expression at the transcriptional level

Wild-type p53 has been shown to repress survivin expression at the mRNA level. Using an adenovirus vector for wild-type p53, human ovarian cancer cell line 2774qw1 (which expresses mutant p53) was transfected. mRNA levels of survivin were analyzed by real-time quantitative PCR (RT-PCR) and showed time-dependent down regulation of survivin mRNA levels when the cells were infected with wild-type p53. A 3.6 fold decrease of survivin mRNA level was observed 16 hours after infection initiation and decreased 6.7 fold 24 hours after infection. Western blot results do show that there is indeed the p53 from the adenoviral vector was being expressed in the cells using antibody specific for p53. The expression of p53 levels indicative of its role in survivin repression shows that p53 started to be expressed 6 hours into infection and had its highest level at 16–24 hours. To further confirm that endogenous wild-type p53 is really causing the repression of survivin gene expression, the authors induced A549 (human lung cancer cell line with wild-type p53) and T47D (human breast cancer cell line with mutant p53) cells with DNA-damaging agent adriamycin to trigger the physiological p53 apoptotic response in these cancer cells and compare the survivin levels measured to the same cells without DNA damage induction. The A549 line, which intrinsically has functioning wild-type p53, showed significant reduction in survivin levels compared to non-induced cells. This same effect was not seen in T47D cells that carry mutant inactive p53.

P53's normal function is to regulate genes that control apoptosis. As survivin is a known inhibitor of apoptosis, it can be implied that p53 repression of survivin is one mechanism by which cells can undergo apoptosis upon induction by apoptotic stimuli or signals. When survivin is over-expressed in the cell lines mentioned in the previous paragraph, apoptotic response from DNA-damaging agent adriamycin decreased in a dose-dependent manner. This suggests that down-regulation of survivin by p53 is important for p53-mediated apoptotic pathway to successfully result in apoptosis. It is known that a defining characteristic of most tumors is the over-expression of survivin and the complete loss of wild-type p53. The evidence put forth by Mirza et al. shows that there exists a link between survivin and p53 that can possibly explain a critical event that contributes to cancer progression.

p53 suppression of survivin expression

In order to see whether p53 re-expression in cancer cells (that have lost p53 expression) has the suppressive effect on the promoter of the survivin gene, a luciferase reporter construct was made. The isolated survivin promoter was placed upstream of the luciferase reporter gene. In a luciferase reporter assay, if the promoter is active, the luciferase gene is transcribed and translated into a product that gives off light that can measured quantitatively and, thus, represents the activity of the promoter. This construct was transfected into cancer cells that had either wild-type or mutant p53. High luciferase activity was measured in the cells with mutant p53 and significantly lower luciferase levels were measured for cells with wild-type p53.

Transfection of different cell types with wild-type p53 was associated with a strong repression of the survivin promoter. Transfection with mutant p53 was not shown to strongly repress the survivin promoter. More luciferase constructs were prepared with varying degrees of deletion from the 5' end of the survivin promoter region. At one point, there was deletion that caused the survivin levels to be indifferent to the presence of the p53 over-expression plasmid, indicating that there is a specific region proximal to the transcription start site that is needed for p53 suppression of survivin. Although it has been found that two p53 binding sites are located on the survivin gene promoter, analysis using deletions and mutations has shown that these sites are not essential to transcriptional inactivation.

Instead, it is observed that modification of the chromatin inside of the promoter region may be responsible for the transcriptional repression of the survivin gene. This is explained below in the epigenetic regulation section.

Cell cycle regulation

Survivin is shown to be clearly regulated by the cell cycle, as its expression is found to be dominant only in the G2/M phase. This regulation exists at the transcriptional level, as there is evidence of the presence of cell-cycle-dependent element/cell-cycle gene homology region (CDE/CHR)boxes located in the survivin promoter region.  Further evidence to support this mechanism of regulation includes the evidence that surivin is poly-ubiquinated and degraded by proteasomes during interphase of the cell cycle.  Moreover, survivin has been shown to localize to components of the mitotic spindle during metaphase and anaphase of mitosis. Physical association between polymerized tubulin and survivin have been shown in vitro as well. It is also shown that post-transcriptional modification of survivin involving the phosphorylation of Thr34 leads to increased protein stability in the G2/M phase of the cell cycle.

It is known from Mirza et al. that repression of survivin by p53 is not a result of any cell cycle progressive regulation. The same experiment by Mirza et al. with regard to determining p53 suppression of survivin at the transcriptional level was repeated, but this time for cells arrested in different stages of the cell cycle. It was shown that, although p53 arrests the numbers of cells to different extents in different phases, the measured level of survivin mRNA and protein levels were the same across all the samples transfected with the wild-type p53. This shows that p53 acts in a cell-cycle independent manner to inhibit survivin expression.

Epigenetic and genetic regulation

As observed through the literature, survivin is found to be over-expressed across many tumour types. Scientists are not sure of the mechanism that causes this abnormal over-expression of survivin; however, p53 is downregulated in almost all cancers, so it is tempting to suggest that survivin over-expression is due to p53 inactivity. Wagner et al. investigated the possible molecular mechanism involved with the over expression of survivin in acute myeloid leukemia (AML). In their experiments, they did both an epigenetic and a genetic analysis of the survivin gene promoter region in AML patients and compared the observations to what was seen in peripheral blood mononuclear cells (PBMCs) that have been shown to express no survivin. Assuming that the molecular mechanism of survivin re-expression in cancerous cells is at the transcriptional level, the authors decided to look at particular parts of the promoter region of survivin in order to see what happens in cancer cells that does not happen in normal cells that causes such a high level of survivin to be expressed. With regards to an epigenetic mechanism of survivin gene regulation, the authors measured the methylation status of the survivin promoter, since it is accepted that methylation of genes plays an important role in carcinogenesis by silencing of certain genes or vice versa. The authors used methylation specific polymerase chain reaction with bisulfite sequencing methods to measure the promoter methylation status in AML and PBMCs and found unmethylated survivin promoters in both groups. This result shows that DNA methylation status is not an important regulator of survivin re-expression during leukemogenesis. However, De Carvalho et al. performed a DNA methylation screening and identified that DNA methylation of IRAK3 plays a key role in survivin up-regulation in different types of Cancer, suggesting that epigenetic mechanisms plays an indirect role on abnormal over-expression of survivin. With regard to genetic analysis of the survivin promoter region, the isolated DNA of AML and PBMCs were treated with bisulfite, and the survivin promoter region sequence was amplified out with PCR and sequenced to look for any particular genetic changes in the DNA sequence between the two groups. Three single-nucleotide polymorphisms (SNPs) were identified and were all present both in AML patients and in healthy donors. This result suggests that the occurrence of these SNPs in the promoter region of the survivin gene also appears to be of no importance to survivin expression. However, it has not been ruled out yet that there may be other possible epigenetic mechanisms that may be responsible for a high level of survivin expression observed in cancer cells and not in normal cells. For example, the acetylation profile of the survivin promoter region can also be looked at. Different cancer and tissue types may have slight or significant differences in the way survivin expression is regulated in the cell, and, thus, the methylation status or genetic differences in the survivin promoter may be observed to be different in different tissues. Thus, further experiments assessing the epigenetic and genetic profile of different tumour types must be investigated.

As a drug target

Expression in cancer as a tool for cancer-directed therapy

Survivin is known to be highly expressed in most tumour cell types and absent in normal cells, making it a good target for cancer therapy. The exploitation of survivin's over-active promoter in most cancer cell types allows for the delivery of therapeutics only in cancer cells and removed from normal cells.

Small interfering RNA (siRNA) are synthetic antisense oligonucleotides to the mRNA of the gene of interest that works to silence the expression of a particular gene by its complementary binding. siRNAs, such as LY2181308, bound to the respective mRNA results in disruption of translation of that particular gene and thus the absence of that protein in the cell. Thus, the use of siRNAs has great potential to be a human therapeutic, as it can target and silence the expression of potentially any protein you want. A problem arises when siRNA expression in a cell cannot be controlled, allowing its constitutive expression to cause toxic side-effects. With regard to practical treatment of cancer, it is required to either deliver the siRNAs specifically into cancer cells or control the siRNA expression. Previous methods of siRNA therapy employ the use of siRNA sequences cloned into vectors under the control of constitutively active promoters. This causes a problem, as this model is non-specific to cancer cells and damages normal cells too. Knowing that survivin is over-expressed specifically in cancer cells and absent in normal cells, one can imply that the survivin promoter is active only in cancer cells. Thus, the exploitation of this difference between cancer cells and normal cells will allow appropriate therapy directed only at the cells in a patient that are harmful. In an experiment to demonstrate this idea, Trang et al. have created a cancer-specific vector expressing siRNA for green fluorescent protein (GFP) under the human survivin promoter. MCF7 breast cancer cells were cotransfected with this vector and a GFP-expressing vector as well. Their major finding was that MCF7 cells transfected with the siRNA vector for GFP under the survivin promoter had a significant reduction in GFP expression then the cells transfected with the siRNA vector under a cancer non-specific promoter. Moreover, normal non-cancerous cells transfected in the same way mentioned above showed no significant reduction in GFP expression. This is implying that, in normal cells, survivin promoter is not active, and, thus, the siRNA will not be expressed under an inactive survivin promoter.

Antisense oligonucleotides targeting survivin mRNA

As it is known that survivin is over-expressed in most cancers, which may be contributing to the cancer cells' resistance to apoptotic stimuli from the environment. The use of antisense survivin therapy hopes to render cancer cells susceptible to apoptosis by eliminating survivin expression in the cancer cells.

Olie et al. developed different 20-mer phosphorothioate antisense oligonucleotides that target different regions in the mRNA of the survivin gene. The antisense function of the oligonucleotides allows binding to surviving mRNA and, depending on the region on which it binds, might inhibit surviving mRNA from being translated into a functional protein. Real-time PCR was used to assess the levels of mRNA present in a lung adenocarcinoma cell line A549 that overexpresses survivin. The best antisense oligonucleotide was identified that effectively down-regulated survivin mRNA levels and resulted in apoptosis of the cells. Survivin’s role in cancer development in the context of a signaling pathway is its ability to inhibit activation of downstream caspase-3 and -7 from apoptosis inducing stimuli. The overexpression of survivin in tumors may serve to increase the tumors resistance to apoptosis and, thus, contribute to cell immortality even in the presence of death stimuli. In this experiment, the oligonucleotide 4003 that targets nucleotides 232-251 of survivin mRNA was found to be the most effective at down-regulating the levels of survivin mRNA in the A549 tumour line. The 4003 oligonucleotides were introduced into the tumour cells by transfection. Further experiments were then conducted on 4003. One of the additional experiments involved determining the dose-dependent effect of 4003 on the down-regulation of survivin mRNA levels. It was found that a concentration of 400 nM resulted in a maximum down-regulation of 70% of the initial survivin mRNA present. Another experiment on 4003 involved assessing any biological or cytotoxic effect 4003 down-regulation of survivin mRNA has on A549 cells using the MTT assay. The numbers of A549 cells transfected with 4003 significantly decreased with increasing concentration of 4003 compared to cells transfected either with a mismatch form of the 4003 or lipofectin control. Many physical observations that confirmed the induction of apoptosis by 4003 were made. For example, lysates of the 4003-treated cells showed increased levels of caspase-3-like protease activity; nuclei were observed to be condensed and chromatin was fragmented.

Cancer immunotherapy

Survivin has been a target of attention in recent years for cancer immunotherapy, as it is an antigen that is expressed mostly in cancer cells and absent in normal cells. This is because survivin is deemed to be a crucial player in tumour survival. There has been much evidence accumulated over the years that shows survivin as a strong T-cell-activating antigen, and clinical trials have already been initiated to prove its usefulness in the clinic.

Activation of the adaptive immune system

A. Cellular T Cell Response

The first evidence of survivin-specific CTL recognition and killing was shown in an assay wherein cytotoxic T cells (CTLs) induced lysis of B cells transfected to present survivin peptides on its surface. The naive CD8+ T cells were primed with dendritic cells and could therefore recognize the specific peptides of survivin presented on the surface Major Histocompatibility Complex I (MHC I) molecules of the B cells.

B. Humoral Antibody Response

Taking blood samples from cancer patients, scientists have found antibodies that are specific for survivin. These antibodies were absent in the blood samples of healthy normal patients. Therefore, this shows that survivin is able to elicit a full humoral immune response. This may prove useful, as one could measure the level of survivin-specific antibodies in the patient's blood as a monitor of tumour progression. In acquiring the humoral response to tumour antigens such as survivin, CD4+ T cells are activated to induce B cells to produce antibodies directed against the particular antigens.

The isolation of the antibodies specific for survivin peptides is useful, as one can look at the structure and sequence of the epitope binding groove of the antibody and, therefore, deduce possible epitopes that may fit in that particular antibody groove.  Therefore, one can determine the particular peptide portion of the survivin protein that is bound most efficiently and most commonly by humoral antibodies generated against survivin. This will lead to the production of more specific survivin vaccines that contain a specific portion of the survivin protein that is known to elicit a good immune response, generate immune memory, and allow for protection from tumour development.

Over-expression in tumours and metastatic tissues

Xiang et al. found a new approach in inhibiting tumour growth and metastasis by simultaneously attacking both the tumour and its vasculature by a cytotoxic T cell (CTL) response against the survivin protein, which will later result in the activation of apoptosis in tumour cells.

The idea and general principle behind his technique is described below. Mice were immunized with the oral vaccination and then subjected to tumour challenges by injecting them in the chest with a certain number of tumour cells and a Matrigel pre-formed extracellular matrix to hold the tumour cells together. The mice were sacrificed and the endothelium tissue was stained with a fluorescent dye that would aid in the quantification of tumour neovascularisation using a Matrigel assay. There was found to be a significant difference between the control and test groups, whereby mice given the vaccine had less angiogenesis from the tumour challenge than the control mice that were not given any of the vaccine prior to tumour challenge. In vitro assays and other tests were also performed to validate the idea of the occurrence of an actual immune response to support what they observed in the mice. For example, the spleen on the challenged mice were isolated and measured for the presence of any cytokines, and specifically activated immune cell groups that would indicative that a specific immune response did occur upon vaccination. The isolated CTLs specific for the survivin protein after vaccination of the mice were used in cytoxicity assays where mice tumour cells expressing survivin were shown to be killed upon incubation with the specific CTLs.

By using an oral DNA vaccine carried in an attenuated non-virulent form of Salmonella typhimurium, which co-encoded secretory chemokine CCL21 and survivin protein in C57BL/6J mice, Xiang et al. have been able to elicit an immune response carried out by dendritic cells (DCs) and CTLs to eliminate and suppress the pulmonary metastases of non-small cell lung carcinoma. The activation of the immune response is most likely taking place in the secondary lymphoid organ called the Peyer’s Patch in the small intestine where DCs take up the survivin protein by phagocytosis and present them on their surface receptors to naive CD8+ T cells (uninactivated CTL) to achieve a specific immune response targeting survivin exclusively. Activated CTLs specific for a particular antigen kill their target cells by first recognizing parts of the survivin protein expressed on MHC I (immunohistocompatability) proteins presented on the surface of tumour cells and vasculature and then releasing granules that induce the tumour cells to undergo apoptosis. The DNA vaccine contained the CCL21 secretory chemokine as a way to enhance the likelihood of eliciting the immune response by better mediating the physical interaction of the antigen-presenting DCs and the naive CD8+ T cells, resulting in a greater likelihood of immune activation.

Resveratrol-mediated sensitization

It has been shown by Fulda et al. that the naturally occurring compound resveratrol (a polyphenol found in grapes and red wine) can be used as a sensitizer for anticancer drug-induced apoptosis by the action of causing cell cycle arrest. This cell cycle arrest causes a dramatic decline in survivin levels in the cells, as it is known from the literature that survivin expression is highly linked with the cell cycle phase state. Thus, the decrease in survivin, which is a contributing factor to chemotherapy resistance and apoptosis induction therapies, would render the cancer cells more prone to such cancer treatments. Fulda et al. have demonstrated the benefits of resveratrol through a series of experiments. First, the authors of the paper tested the intrinsic cytotoxic effects of resveratrol. They found that it induced moderate apoptosis levels only in SHEP neuroblastoma cells. After, they tested resveratrol in combination with several different known anticancer agents. They found a consistent increase in the level of apoptosis induced by the drugs when resveratrol was also present. Moreover, they varied the order with which either the drugs or resveratrol was introduced to the cancer cells to determine whether the sequence of treatment had any important effect. It was found that the highest levels of apoptosis induction were observed when resveratrol was added prior to anticancer drug treatment. Next, the authors tested for any differential sensitivity to apoptosis linked to the phase of the cell cycle the cells were in. Analysis by flow cytometry revealed an accumulation of cells in S phase upon treatment with resveratrol. The cells were also halted in different phases of the cell cycle using special compounds and then treated with the anticancer drugs. They found that cells halted in S phase were significantly more sensitive to the cytotoxic effects of the drugs.

To determine the involvement of survivin in resveratrol-mediated sensitization, the authors decided to test whether downregulation of the specific survivin protein expression would confer a similar effect on the phenotype of resveratrol-treated cells. In terms of seeing at which level resveratrol worked, they did a northern blot and found that resveratrol treatment resulted in a decrease in survivin mRNA levels, thus implying resveratrol’s inhibitory action at the transcriptional level. To further see whether survivin played a key role in sensitization of the cancer cells to cytotoxic drugs, survivin antisense oligonucleotides were used to knock down any survivin mRNA, and, thus, its possibility to be translated is also eliminated. siRNAs for survivin are complements in sequence to the mRNA sequence encoding survivin. When these siRNAs for survivin are introduced into cells, they will bind to the respective complementary mRNA and, thus, prevent its translation since the mRNA is now impeded from proper physical interaction with the translational machinery. In this way, the siRNAs for survivin effectively downregulates survivin expression level in the cell. Cells treated with antisense oligonucleotides for survivin showed similar sensitization to cytotoxic drugs as cells treated with resveratrol, which offers support for the mechanism of action of resveratrol.

Prostate cancer

It has been observed that the development of hormone resistance in prostate cancer may be due to the upregulation of antiapoptotic genes, one of which is survivin.

Zhang et al. hypothesize that, if survivin is a significant contributor to the development of hormonal therapy resistance in prostate cancer cells, targeting survivin and blocking it would enhance prostate cancer cell susceptibility to anti-androgen therapy. The basis of anti-androgen therapy involves using drugs that eliminate the presence of androgens in the cell and cellular environment, since the presence of androgens are known to enhance tumour immortality in prostate cancer cells. Zhang et al. first assessed the level of survivin expression of LNCaP (an androgen-dependent prostate cancer cell line that expresses intact androgen receptors) using quantitative Western analysis and found high expression of survivin in these cells. Cells exposed to dihydrotestosterone (DHT), an exogenous androgen, showed increased levels of survivin expression only and not other IAP family members. This result implicates that androgens in the physiological context may upregulate survivin, which contributes to the resistance of apoptosis observed in the tumour cells. Next, with the addition of Flutamide(an antiandrogen) to the cells, survivin levels were observed to significantly decrease. The LNCaP cells were transduced separately with the different constructs of the survivin gene (mutant or wild-type) and subjected to Flutamide treatment and assessed for the apoptosis level. Flutamide-treated survivin mutant-transduced cells were shown to significantly increase apoptosis by double that of Flutamide treatment alone. On the other end, overexpression of the wild-type survivin was found to significantly reduce the apoptosis levels from Flutamide treatment compared to Flutamide treatment alone. Therefore, these results support the hypothesis that asserts that survivin does play a role in contributing the anti-apoptotic nature of the LNCaP cancer cell line and that inhibiting survivin in prostate cancer cells does appear to enhance the therapeutic effect of Flutamide.

Interactions

Survivin has been shown to interact with:

 Aurora B kinase,
 CDCA8,
 Caspase 3,
 Caspase 7,
 Diablo homolog  and
 INCENP.


Stradouň

Stradouň is a small village in the Ústí nad Orlicí District (before 1 January 2007 in the Chrudim District), Pardubice Region, in the Czech Republic. It had around 170 inhabitants at the last census.

External links

 Short official information about the village (in Czech)


New York State Route 31E

New York State Route 31E (NY 31E) is a  state highway located in western New York in the United States. It serves as a northerly alternate route of NY 31 between the village of Middleport in eastern Niagara County and the nearby village of Medina in Orleans County. NY 31E connects to NY 31 at each end by way of a short overlap with NY 271 in Middleport. Most of NY 31E parallels the Erie Canal, and parts of the route run adjacent to the waterway. Outside of the two villages at each end, NY 31E traverses mostly rural areas of the towns of Royalton and Ridgeway.

The origins of the route date back to 1914 when its routing was included as part of a spur route of Route 30, a cross-state, unsigned legislative route. The spur route was eliminated in 1921, and the road went unnumbered until 1930 renumbering of state highways in New York when it became part of NY 3. Most of NY 3 west of Rochester was replaced by NY 31 in the mid-1930s, and NY 31 was realigned onto its present, more southerly alignment between Middleport and Medina in 1949. Its former canalside alignment became NY 31E at this time. Most of NY 31E is maintained by the New York State Department of Transportation (NYSDOT); however, two sections in Niagara County and Medina are locally maintained.

Route description

NY 31E begins at an intersection with NY 31 in the village of Middleport in eastern Niagara County. The route heads north as Main Street, overlapping with NY 271 (which also begins at NY 31) through the mostly residential village. Just south of the Erie Canal, NY 31E and NY 271 enter Middleport's small business district, centered on the junction of Main and State Streets. Here, NY 31E and NY 271 split, with NY 31E following State Street eastward through the eastern portion of the community. Along this stretch, the route serves Royalton–Hartland High School and Royalton-Hartland Middle School before exiting Middleport and entering a slightly more open area of the town of Royalton. NY 31E runs past undeveloped land to the north and a smattering of homes to the south on its way into the Orleans County town of Shelby New York|Ridgeway]].

East of the county line, NY 31E becomes Telegraph Road and continues across increasingly less developed areas of Ridgeway. The Erie Canal is visible from most of this section of NY 31E, and about  of the highway runs directly alongside the waterway, which curves several times between Middleport and Medina. The two separate at the small hamlet of Shelby Basin, where the route follows a more southerly alignment into a more populated area. Not far to the east is the village of Medina, where NY 31E changes names to West Center Street. The route heads east across the residential western half of the village, passing Prospect Avenue (former NY 63) on its way into Medina's business district. NY 31E passes the village's post office and enter the Main Street Historic District, where NY 31E terminates at NY 31 and NY 63.

Maintenance along the route varies by location. From the Niagara–Orleans county line to the Medina village line, NY 31E is maintained by NYSDOT. All of the route within Niagara County, excluding the state-maintained overlap with NY 271 in Middleport, is county-maintained as the unsigned County Route 143 (CR 143). In Medina, NY 31E is entirely maintained by the village, though between the village line and Prospect Avenue (former NY 63), it was NYSDOT-maintained for  to its terminus at NY 31 until 2010.

History
In 1914, the New York State Legislature added two spur routes to Route 30, an unsigned legislative route extending from Niagara Falls in western Niagara County to Rouses Point in Clinton County via Medina. The first of these began at Route 30 (modern NY 63) and followed what is now NY 31E west to Middleport, from where it continued toward Lockport on current NY 31. On March 1, 1921, most of Route 30's Medina–Lockport spur route was supplanted by an extended Route 20. The lone exception was from Medina to Middleport, where Route 20 utilized modern NY 31 instead. When the first set of posted routes in New York were assigned in 1924, all of legislative Route 20 west of Rochester became part of NY 3.

NY 3 remained on what is now NY 31 between Middleport and Medina until the 1930 renumbering of state highways in New York when it was realigned between the two villages to use what is now NY 31E. U.S. Route 104 was assigned to all of Ridge Road , replacing then-NY 31 west of Rochester. As a result, NY 31 was shifted southward onto most of NY 3 from Niagara Falls to Rochester, including between Middleport and Medina. NY 31 was realigned on January 1, 1949, to follow its current alignment between the two villages. The former routing of NY 31 was redesignated as NY 31E, a northerly alternate route of NY 31.

The alignment of NY 31E has not changed since that time; however, ownership of parts of the route has changed hands over the years. Until 1998, NY 31E was state-maintained from its western terminus in Middleport to the Medina village line. On October 1, 1998, ownership and maintenance of NY 31E between NY 271 in Middleport and the Niagara–Orleans county line was transferred from the state to Niagara County as part of a highway maintenance swap between the two levels of government. This section of the route was subsequently co-designated, but not signed, as CR 143 by Niagara County.

Major intersections


Miguel Bombarda

Miguel Augusto Bombarda (6 March 1851 – 3 October 1910) was a Portuguese physician, psychiatrist and politician. He is perhaps most widely remembered as one of the major conspirators of the 5 October 1910 revolution, although he was shot and killed the day before the coup took place by one of his patients.

Selected publications

Contribuição para o estudo dos microcephalus (1894)
Lições sobre a epilepsia e as pseudo-epilepsies (1896)
Estudos Biológicos. A Consciência e o Livre Arbítrio (1898)
A sciencia e o Jesuitismo: replica a um padre sabio (1900)


Jim McGarr

James Vincent McGarr (November 9, 1888 – July 21, 1981), nicknamed "Reds" was a Major League Baseball replacement player on May 18, 1912 when the Detroit Tigers went on strike to protest the suspension of Ty Cobb. Born in Philadelphia, McGarr played second base for the Tigers during the one-game strike, going went hitless in four at-bats. In the field, he recorded one putout, three assists, and one error. McGarr and fellow replacement player Dan McGarvey were friends who had also been teammates at Georgetown College.  Other replacement players that day included Allan Travers, Hap Ward, Billy Maharg, Bill Leinhauser, Vincent Maney, and Jack Smith.

McGarr was the last of the "strikebreakers" to die, in Miami, Florida at age 92 in 1981; ironically, when McGarr died, the major leagues were on strike.

External links

 SABR Biography of Replacement Player
 The Suspension Game


Ding'an Road station

Ding'an Road () is a station on Line 1 of the Hangzhou Metro in China. It was opened in November 2012, together with the rest of the stations on Line 1. It is located in the Shangcheng District of Hangzhou.


Andries Bekker

Andries Bekker (born 5 December 1983 in Cape Town, South Africa) is a South African rugby union player for the Springbok rugby team as well as Kobelco Steelers in the Japanese Top League.

Playing career

Bekker made his South African debut in 2008 against Wales in a 43–17 victory in Bloemfontein.

At 6'11" he is one of the tallest international rugby players, but he only made the Springbok side regularly after the retirement of locks Bakkies Botha and Victor Matfield. He is the son of former Springbok International Hennie Bekker who was also a lock.

Holding the record for being the tallest Springbok in history, Andries Bekker was an imposing figure in the DHL Stormers lineout. Despite his large physique, he is blessed with mobility and pace; aspects which have seen him become an integral part of the current Springbok squad. His experience and all-round ability was to be crucial to the success of a relatively young pack in 2010.

In 2013 Bekker agreed to a move to Japanese club Kobelco Steelers.

Accolades

Bekker was a Vodacom Super 14 Rugby Player of the Year nominee in 2008.


Moderna de Pueblo

Raquel Córcoles (born December 31, 1986), better known as Moderna de Pueblo, is a Spanish illustrator and author of comics.

Biography
Corcoles studied advertising, journalism and audiovisual communication at Rovira i Virgili University in Tarragona and has a degree in journalism from King Juan Carlos University in Madrid. She has been editor of InformativosTelecinco.com in charge of economic information. Corcoles started as a comic book illustrator in August 2011 in the magazine El Jueves, where she published a weekly comic page, and that same year she began to publish a weekly comic strip in the Cuore magazine. She also collaborates with other media such as GQ magazine and the newspaper El País.

Moderna de Pueblo
The name of Moderna de Pueblo ("Modern Villager") comes from a character created in 2010 in her blog. This character became a viral phenomenon with more than 200,000 followers on Facebook. Her comics are about a twenty-year-old blonde girl who always wear sunglasses and who moves out of town to succeed in the city. There are other characters stereotypical of urban culture, such as women with everyday problems, frustrated young people, interns and hipsters.

Bibliography


Walter Fremont

Walter Gilbert Fremont, Jr. (July 20, 1924 – January 7, 2007) was dean of the School of Education, Bob Jones University (1953–1990) and “a seminal force in the inauguration and development of the Christian school movement.”

Youth and Education

Fremont was born in Terre Haute, Indiana but was largely reared in Wilmette, Illinois and Southern Hills, a suburb of Dayton, Ohio.  A child of the Depression, Fremont remembered his family having a maid before the crash but afterward having too little money to buy coal.  Eventually the senior Fremont scrabbled back to economic prosperity and by doing so helped to inculcate in his son a belief in persistence, that “once you start something, you don't quit.”

As a child, Fremont was popular, athletic, and mechanically inclined. During World War II, he was drafted and assigned by the Army to study mechanical engineering at Carnegie Institute of Technology.  He then served as an engineering instructor and later as the supervisor of a mobile machine shop in Europe.

Following his discharge, Fremont earned an education degree from the University of Dayton (1947) and a master of science in curriculum development from the University of Wisconsin (1949).  In 1947 he married Gertrude Reed, a nursing student; they had three children.

Teacher and administrator

Fremont became an evangelical Christian in 1941. Even as a teenager, he made an interdenominational Bible study the focus of his spiritual and social life.  In 1950, with one year of GI Bill benefits remaining, Fremont decided to study Bible at fundamentalist Bob Jones University in Greenville, South Carolina, because of the University's reputation for “instilling soul-winning fervor” into its students.  Fremont was immediately asked to teach educational psychology and taught both semesters while taking thirty hours of Bible courses.

The following year he became a full-time member of the education faculty eventually teaching courses in psychology, counseling, and educational administration.  Fremont was a popular teacher, humorous, dramatic, acrobatic, and wildly enthusiastic. Meanwhile, he also taught Sunday school, engaged in street preaching, and held Bible studies at a local high school.

In 1953, during a period of upheaval at BJU, Fremont was appointed Dean of the School of Education.  He was 29 and served as dean for thirty-seven years. From 1953 to 1959, Fremont worked on his doctorate at Pennsylvania State University during the summers. He completed his dissertation in about three months by writing from six in the evening until two in the morning.  Fremont eventually helped expand the “meager offerings” of the BJU education department at his arrival into full-scale elementary, secondary, and graduate programs.

Proponent of Christian education

Fremont's dissertation outlined principles of administration in evangelical Protestant Christian schools, a tiny segment of the educational world in the 1950s but his burden for the rest of his career.  Fremont became an early mainstay of BJU Press in a successful effort to make Bob Jones University a leader in the new Christian school movement.  Bob Jones III called Fremont "the John Dewey of Christian primary and secondary education," and Keith Wiebe, president of the American Association of Christian Schools referred to him as a man with a "contagious passion for Christian education." Fremont's successor as dean estimated that during Fremont's tenure the percentage of BJU education graduates who entered Christian school teaching rose from 5% to 90%.

Fremont differentiated traditional education, with its emphasis on rote memory, from Christian education, which he believed should emphasize Bible principles.  Arlin Horton, founder of Pensacola Christian College, accused Fremont of promoting progressive education, and although Horton and Fremont remained personal friends, their educational philosophies diverged during the 1970s, a separation mirrored in the textbooks published by BJU Press and Horton's A Beka Book.

Fremont helped found a Children's Gospel Club in Greenville and served on the executive committee of a Greenville-based mission board.  But he claimed that his most effective ministry occurred in camp settings, believing that the camping environment—one that brought people into God's creation and separated them from normal distractions—was the best place for communicating spiritual truth.  In 1967, he and longtime friend, Ken Hay helped found The Wilds with Carl Blythe and Joe Henson.  Fremont served on the board of directors for twenty-eight years and often spoke at couple's retreats, which were always completely booked.

Illness and death

In 1986, Fremont was diagnosed with amyotrophic lateral sclerosis (Lou Gehrig's disease) and given two to five years to live.  He continued to serve as dean until 1990, taught from a wheelchair for one more year, and then survived fifteen years more, co-authoring several books from his hospital bed.  The Wilds dedicated a Fremont Inn in 1992, and Bob Jones University named the Fremont Fitness Center in his honor in 1993.  In January 2007, Fremont died less than a hundred yards away from the Fitness Center.

Books

Formula for Family Unity: A Practical Guide for Christian Families (1980) (with Trudy Fremont) .
Forming a New Generation: A Practical Guide for Youth Leaders (1990) (with Trudy and Gilbert Fremont)
Becoming an Effective Christian Counselor (1996) (with Trudy Fremont)
Rambunctious Rattler (1999) (with Susan W. Young), juvenile fiction
Power to Serve: Living a Joyful Spirit-filled Life (2002) (with Carson K. Fremont)


Chvátal graph

In the mathematical field of graph theory, the Chvátal graph is an undirected graph with 12 vertices and 24 edges, discovered by .

It is triangle-free: its girth (the length of its shortest cycle) is four. It is 4-regular: each vertex has exactly four neighbors. And its chromatic number is 4: it can be colored using four colors, but not using only three. It is, as Chvátal observes, the smallest possible 4-chromatic 4-regular triangle-free graph; the only smaller 4-chromatic triangle-free graph is the Grötzsch graph, which has 11 vertices but has maximum degree 5 and is not regular.

This graph is not vertex-transitive: the automorphisms group has one orbit on vertices of size 8, and one of size 4.

By Brooks’ theorem, every k-regular graph (except for odd cycles and cliques) has chromatic number at most k. It was also known since  that, for every k and l there exist k-chromatic graphs with girth l. In connection with these two results and several examples including the Chvátal graph,
 conjectured that for every k and l there exist k-chromatic k-regular graphs with girth l. The Chvátal graph solves the case k = l = 4 of this conjecture. Grünbaum's conjecture was disproven for sufficiently large k by Johannsen (see ), who showed that the chromatic number of a triangle-free graph is O(Δ/log Δ) where Δ is the maximum vertex degree and the O introduces big O notation. However, despite this disproof, it remains of interest to find examples such as the Chvátal graph of high-girth k-chromatic k-regular graphs for small values of k.

An alternative conjecture of  states that high-degree triangle-free graphs must have significantly smaller chromatic number than their degree, and more generally that a graph with maximum degree Δ and maximum clique size ω must have chromatic number

The case ω = 2 of this conjecture follows, for sufficiently large Δ, from Johanssen's result. The Chvátal graph shows that the rounding up in Reed's conjecture is necessary, because for the Chvátal graph, (Δ + ω + 1)/2 = 7/2, a number that is less than the chromatic number but that becomes equal to the chromatic number when rounded up.

The Chvátal graph is Hamiltonian, and plays a key role in a proof by  that it is NP-complete to determine whether a triangle-free Hamiltonian graph is 3-colorable.

The characteristic polynomial of the Chvátal graph is . The Tutte polynomial of the Chvátal graph has been computed by .

The independence number of this graph is 4.

Gallery


Enid Gordon-Gallien

Enid Gordon-Gallien (c. 1887 -18 June 1931), was a British adventurer and pilot who was awarded the Back Award in 1930 for her expedition in Tanganyika.

Life
Enid Gordon-Gallien was the wife of Captain Gordon Gallien, an engineer of the Anglo-Persian Oil Company. With him she had travelled extensively. She had been involved in the First World War in both France and Egypt. Gordon-Gallien was a member of the Royal Geographical Society. She studied with Mr Reeves of the Royal Geographical Society in 1925 and in 1928 and she studied field astronomy and surveying. At that point she begin investigating what expeditions she could run. The president of the society, Colonel Sir Charles Close, proposed the as then unmapped Kalambo Falls. Prior to this, Gordon-Gallien had driven her car across the desert to Baghdad as well as becoming shipwrecked on the Barrier Reef while journeying to join her husband in Borneo.

Gordon-Gallien began at Dar-es-Salaam on 15 June 1928. Other members of the party were J. W. Cornwall, a surveyor, and Colin Rose, a geologist. The expedition was written up and presented to the society in 1929. As a result of her work on the expedition, Gordon-Gallien was awarded the Back Award in 1930.

Gordon-Gallien gained her aviators certificate in August 1930 through National Flying Services at Hanworth Aerodrome. However, she was killed in a plane crash, as a passenger, when flying with her friend and co-owner of the plane, the noted pilot Sicele O'Brien on 18 June 1931.

References and sources


HTMS Chakri Naruebet

HTMS Chakri Naruebet (CVH-911) (, meaning 'Sovereign of the Chakri Dynasty', the Thai monarchy's ruling family) is the flagship of the Royal Thai Navy (RTN), and Thailand's first and only aircraft carrier, although the RTN refers to her as an "Offshore Patrol Helicopter Carrier". Based on the Spanish Navy's  design and constructed by Spanish shipbuilder Bazán, Chakri Naruebet was ordered in 1992, laid down in 1994, launched in 1996, and commissioned into the RTN in 1997. The ship is the smallest functioning aircraft carrier in the world.

The aircraft carrier was designed to operate an air group of V/STOL fighter aircraft and helicopters, and is fitted with an aircraft ski-jump. Initial intentions were to operate a mixed air group of ex-Spanish AV-8S Matador Harrier V/STOL aircraft and Sikorsky SH-60 Seahawk helicopters. However, by 1999 it was reported that only one Matador was operational due to parts, training, and fiscal limitations, although three Matadors were spotted on the ship during a show of force after the 29 January 2003 Phnom Penh riots in Cambodia. Her entire Harrier V/STOL jet fleet was removed from service in 2006. Although Chakri Naruebet was intended for patrols and force projection in Thai waters, a lack of funding brought on by the 1997 Asian Financial Crisis meant the carrier has spent most of her career docked at the Sattahip naval base.

Chakri Naruebet has been deployed on several disaster relief operations, including in the aftermath of the 2004 Indian Ocean earthquake and tsunami, and in response to separate flooding incidents in late-2010 and early-2011. Other than disaster relief, the carrier's few other departures from port are for a single training day per month, and transportation of the Royal Family of Thailand, leading to claims by some naval commentators that the ship is merely an oversized royal yacht.

Design
The original plan was to acquire a 7,800 ton vessel from Bremer Vulcan, but the Thai government cancelled this contract on 22 July 1991. A new contract for a larger warship to be constructed at Bazán's shipyard in Ferrol, Spain was signed by the Thai and Spanish governments on 27 March 1992. The proposed vessel was based on the design of the Spanish Navy aircraft carrier , which in turn was based on the United States Navy's Sea Control Ship concept. The design is formally referred to by the RTN as an "Offshore Patrol Helicopter Carrier".

Chakri Naruebet while still operating with Harriers was the smallest aircraft carrier with a fixed wing air group in operation in the world. She displaces 11,486 tons at full load. The carrier is  long between perpendiculars, and  overall. She is  wide at the waterline, with a maximum beam of , and a draught of . The warship has a ship's company of 62 officers, 393 sailors, and 146 aircrew. Up to 675 additional personnel can be transported, usually from the Royal Thai Marine Corps.

Chakri Naruebet is propelled by a combined diesel or gas (CODOG) system. Each of the two, five-bladed propellers is connected to a Bazán-MTU 16V1163 TB83 diesel engine (providing , used for cruising speed), and a General Electric LM2500 gas turbine (providing , used to reach top speed for short periods). Chakri Naruebet has a maximum speed of , although she can only reach  with the diesels alone. She has a maximum range of  with a constant speed of , and  at . There are provisions to install an SPS-64 surface search radar and a hull-mounted sonar, but neither has been fitted as of 2008. Fire control facilities are also yet to be fitted.

The carrier is equipped with four SRBOC decoy launchers, and an SLQ-25 towed decoy.

In April 2012 Saab won a contract to upgrade Chakri Naruebets command and control systems. This will include fitting a 9LV Mk4 command and control system to the ship as well as a Sea Giraffe AMB radar and improved datalinks.

Construction

Work on the Thai carrier commenced in October 1993, although it was not until 12 July 1994 that the hull was laid down. Chakri Naruebet was launched on 20 January 1996 by Queen Sirikit.

Sea trials were conducted from October 1996 to January 1997, the latter part of which was in coordination with the Spanish Navy. This was followed by aviation trials at Rota, Spain. The carrier was handed over on 27 March 1997, when she was commissioned into the RTN. She arrived in Phuket on 4 August 1997 following a 42-day voyage from Spain, and formally entered service on 10 August.

Chakri Naruebet cost US$336 million to build.

Role and operational history
Chakri Naruebet is the first aircraft carrier to be operated by a Southeast Asian nation. She is assigned to the Third Naval Area Command, and her intended duties include operational support of the RTN's amphibious warfare forces, patrols and force projection around Thailand's coastline and economic exclusion zone, disaster relief and humanitarian missions, and search-and-rescue (SAR) operations. However, at the time the carrier entered service, the 1997 Asian Financial Crisis prevented the necessary funding to operate the ship from being available. Consequently, Chakri Naruebet was usually only operational for a single day per month for training, with the rest of the time spent alongside as a "part-time tourist attraction". The struggles continued for years, but today it is increasingly common to see her participate in maritime exercise or joint forces exercise — including PASSEX, CARAT, Guardian Sea and Cobra Gold. She was also sent for a fleet review such as 2017 International Maritime Review in Singapore.

Naval commentators usually consider Chakri Naruebet to be less an aircraft carrier and more the world's largest and most expensive royal yacht, while the Thai media have nicknamed the ship "Thai-tanic", and consider her to be a white elephant.

Between 4 and 7 November 1997, Chakri Naruebet participated in disaster relief operations following the passage of Tropical Storm Linda across the Gulf of Thailand and the Kra Isthmus. The carrier's main task was to search for and assist any fishing vessels affected by the storm.

Flooding in the Songkhla Province resulted in the carrier's mobilisation in late November 2000. Chakri Naruebet was anchored at an island marina off Songkhla, and used as a base for helicopters and small boats transporting food, supplies, and wounded.

In January 2003 after the burning of Royal Thai Embassy in Cambodia, she was sent for her only "show of force" type mission to date—as an "insurance" policy for  the Pochentong-1 evacuation plan. In the event that the situation escalated and the operation were to be compromised, she would have been able to support Pochentong-2 or a "Siege of Phnom Penh" plan with her Harriers which some of them were still operational at that time. All other missions are exercise or humanitarian-focused.

Following an undersea earthquake in the Indian Ocean, tsunamis struck multiple regions around the Indian Ocean, including the Andaman Sea coast of Thailand. The personnel of Chakri Naruebet were part of a 760-strong response by the Thai military to the disaster. This task force was involved in search-and-rescue around Phuket and the Phi Phi Islands, treatment of wounded and handling of dead, and repair work to schools and government facilities.

During the August 2005 filming of Rescue Dawn, a dramatized biographical film of US Navy pilot Dieter Dengler and his capture during the Vietnam War, the flight deck of Chakri Naruebet was used to represent the carrier .

In November 2010, the ship was involved in flood relief operations following the 2010 Thai floods; anchored off Songkhla Province, relief supplies and food were airlifted to people in the region, while hospital patients were evacuated by the ship's helicopters. Chakri Naruebet was sent to Ko Tao in late-March during the 2011 Southern Thailand floods, as the heavy storms causing the flooding had isolated the island, requiring the evacuation of tourists and local citizens.