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Keir Starmer is under police investigation over claims he broke lockdown rules by having a beer and takeaway with staff during election campaigning last year, after Durham constabulary said they had received “significant new information” about the gathering.
While Labor said it was happy to cooperate with Durham police and insisted no rules were broken, the news is deeply uncomfortable for the party leader, who had called for Boris Johnson to resign when he was investigated for a breach of the law.
Following significant pressure from some newspapers and Conservative MPs to look again at events in Durham, the force said in a statement it had opened an inquiry. The announcement was timed to follow the end of local election campaigning and voting, it said.
“Earlier this year, Durham constabulary carried out an assessment as to whether Covid-19 regulations had been breached at a gathering in Durham City on 30 April 2021,” the statement said.
“At that time, it was concluded that no offense had been established and therefore no further action would be taken. Following the receipt of significant new information over recent days, Durham Constabulary has reviewed that position and now, following the conclusion of the pre-election period, we can confirm that an investigation into potential breaches of Covid regulations relating to this gathering is now being conducted .”
It remains unclear what new information has been given to the police. The local Labor party organized a quiz for activists the same evening but Labor sources said this was held on Zoom and Starmer had nothing to do with the event.
Other recent newspaper coverage has focused on the amount of takeaway food ordered and Labour’s admission that it mistakenly said Angela Rayner, the deputy leader, was not at the event.
A Labor party spokesperson said: “We’re obviously happy to answer any questions there are and we remain clear that no rules were broken.”
One complication for Starmer is that the Labor leader not only called for Johnson to step down as prime minister after he was fined for breaking lockdown rules, but did so as soon as the Metropolitan police began investigating alleged breaches in Downing Street, saying the prime minister should “do the decent thing and resign”.
Asked on Friday about Starmer’s previous comments, the international trade secretary, Emily Thornberry, told the BBC the resignation call had been because Johnson had misled parliament about the parties and thus the situations were different.
The Durham police inquiry will examine, among other information, video footage showing Starmer holding a bottle of beer inside the constituency office of the City of Durham MP, Mary Foy, during the byelection campaign for the nearby Hartlepool seat.
Campaigning was allowed at the time, and the investigation will focus on whether the gathering was reasonably necessary for work-based purposes, as was set out in Covid regulations.
Durham police said in 2020, responding to anger over Dominic Cummings’ travels to the county, that they did not issue fines retrospectively. The Met, which covers London, has been issuing fixed-penalty notices for past breaches against Johnson and others.
In addition to Johnson, the chancellor, Rishi Sunak, and dozens of officials have been fined for breaking Covid rules, and could potentially face more penalties over other events.
In a TV clip on Friday, Starmer said he was “confident there was no breach of the rules”. “I was working, I stopped for something to eat. No party, no breach of the rules,” he said. “The police obviously have got their job to do, we should let them get on with it, but I’m confident that no rules were broken.”
Thornberry said Labor would “answer whatever questions are put to us and we’ll do everything that we can to help”.
“But we’re completely confident that no rules have been broken,” she added. “It’s fine. I appreciate they’ve been put under a lot of pressure – there’s lots of people who’ve been campaigning for the police to open this investigation; lots of Conservative MPs have been asking about it; lots of the Conservative-supporting newspapers have been making a big fuss about it.”
Richard Holden, the Tory MP for North West Durham, who has been among those leading calls for a police inquiry, said the force was doing “exactly the right thing”. He said: “It’s vital that the man who wants to be prime minister is held to the same standard as the prime minister and everybody else.”
Labour’s defense has been that the takeaway was necessary for campaign staff, who carried on working while eating. However, reports have questioned the volume of food and alcohol supplied as well as Labour’s claim that there were no other options for dinner.
Starmer said the food had been ordered as the staff prepared an online event for members. “In Durham, all restaurants and pubs were closed, so takeaways really were the only way you could eat. So this was brought in and at various points people went through to the kitchen and had something to eat, and got on with their work,” he said.
George Holan is chief editor at Plainsmen Post and has articles published in many notable publications in the last decade.

domain: sociology


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Open Access This article is
- freely available
Cancers 2018, 10(7), 231; https://doi.org/10.3390/cancers10070231
Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency
Transgene SA, 400 Bld Gonthier d’Andernach, 67400 Illkirch-Graffenstaden, France
Correspondence: [email protected]
These authors contributed equally to this work.
Current address: Polyplus-Transfection SA, 850 Bld Sébastien Brant, 67400 Illkirch-Graffenstaden, France.
Received: 2 May 2018 / Accepted: 1 June 2018 / Published: 10 July 2018
Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.
Keywords:vaccinia virus; directed evolution; genome shuffling; deVV5-fcu1
The field of oncolytic virotherapy has gained considerable importance in the armamentarium of novel cancer therapies in recent years. In addition to their direct oncolytic activity oncolytic viruses (OVs) are increasingly recognized as an immunotherapy by inducing and amplifying host cell immune responses, leading to immunogenic cell death and long-lasting anti-tumor immunity [1,2,3]. Many different OVs have entered into clinical use. ImlygicTM (talimogene laherparevec, also formerly known as T-VEC), an oncolytic herpes virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), was the first OV to gain approval from the U.S. Food and Drug Administration, and European Medicines Agency in 2015, for the loco-regional treatment of melanoma lesions in the skin and lymph nodes . Many different approaches have been employed to improve the oncolytic potency of OVs, including combination therapies with standard and emerging anticancer therapies . Combination of OVs with immune checkpoint inhibitors (ICI) has also demonstrated synergy . Alternative approaches to improve the efficacy of OVs include the arming of OVs with either therapeutic transgenes or immune stimulatory genes .
Vaccinia virus (VACV) has been the most commonly used poxvirus vector for cancer therapy due to its excellent lytic activity, the capacity to infect a wide range of tumors and the ability to engineer its genome . Moreover, it was shown to display natural tumor tropism, selectively targeting tumors after systemic administration . Several strains of VACV are currently evaluated in preclinical and clinical trials including Wyeth, Western Reserve, Copenhagen and Lister strains [10,11,12,13]. VACV derived OVs have shown good safety profiles but have demonstrated limited therapeutic efficacy as monotherapy. Therefore, new approaches to generate highly potent OVs are needed.
A first approach consists to adapt OVs by serial passages on tumor cells. Using this method, Yoo et al. have selected an improved oncolytic VACV by repeated selective replication in cancerous tissues . A second approach, based on homologous recombination and using the high DNA homology between viruses of the same family, enables the generation of potent OVs. Homologous recombination is the basis for many widely used genetic techniques in virus research, including construction of recombinant vectors . The feasibility of intermolecular recombination between different VACV strains to generate viable hybrids was reported some decades ago [16,17], and it was recently shown that this mechanism could induce multiple genetic exchanges even after one round of selection . Directed evolution is based on extensive intermolecular recombination, and is a method to mimic, and accelerate the process of natural selection to evolve virus under controlled culture conditions. The method was employed to create novel AAV gene delivery vectors , was also used by Kuhn and colleagues to improve the therapeutic index of oncolytic adenoviruses . By pooling different Adenovirus (Ad) serotypes, and by passing them on human tumor cell lines to promote recombination and selection of potent viral variants, they selected ColoAd1 for its rapid and potent replication in colon cancer cells . Recently, they also isolated a new chimeric adenovirus, named OvAd1, on 3D ovarian cell culture . Using the same homologous recombination approach, an improved OV was selected by pooling different orthopoxviruses . Based on these different results, we hypothesized that it should be possible to create new recombinant VACV variants with improved oncolytic properties.
Our starting pool consisted of a mix of three replicative VACV strains, Copenhagen, Western Reserve, and Wyeth, and of the non-propagative Modified Vaccinia of Ankara (MVA) strain. The rationale for choosing these four strains includes the fact they all have been employed in the smallpox vaccination campaign demonstrating a high safety profile , and/or have been extensively studied as viral vectors in both preclinical and clinical studies. Pexa-Vec (pexastimogene devacirepvec, JX-594), the most advanced VACV oncolytic product is a Wyeth (WY) engineered virus to express GM-CSF . Pexa-Vec has now entered a randomized controlled Phase 3 trial in advanced first-line hepato-carcinoma (HCC) with the standard of care, sorafenib (National Clinical Trial NCT02562755). The safety, systemic spread and anti-tumor activity of a Western Reserve (WR)-based VACV has been recently demonstrated in a Phase 1 trial . We used the Copenhagen stain (COP) to engineer a TK-deleted VACV expressing the FCU1 fusion suicide gene for targeted prodrug therapy . It displayed a highly potent anti-tumor effect both in vitro and in vivo, and TG6002, a derivative of this virus containing a second deletion for ribonucleotide reductase , has entered into clinical development in recurrent glioblastoma patients (National Clinical Trial NCT03294486). Modified Vaccinia of Ankara (MVA) is a highly attenuated, non-replicative VACV strain, generated by passaging the VACV Ankara strain more than 570 times in primary chicken embryonic fibroblasts (CEF), thereby losing the ability to produce infectious progeny virus in almost all mammalian cell lines, including human cells . After being used intensively in the smallpox vaccination campaign, MVA has been widely studied as vaccine vector for infectious diseases and cancer . A MVA armed with the suicide gene FCU1 (TG4023)  has been evaluated in a Phase I trial in primary or metastatic liver tumors  and a MVA armed with the tumor-associated antigen MUC1 (TG4010) has been studied in a randomized controlled Phase 2b trial in non-small cell lung cancer, in combination with chemotherapy .
Through this directed evolution process, we selected a VACV hybrid, named deVV5, with enhanced oncolytic properties in a series of human cancer cell lines representing many human solid tumor types. In addition, deVV5 was further modified by inserting the FCU1 gene  into the TK locus, under the control of the strong VACV promoter p11k7.5. The recombinant deVV5-fcu1 virus displayed further increased replicative and oncolytic activity on various human tumor cells. This study demonstrates that, through shuffling of various VACV strains, novel OVs can be generated with improved oncolytic properties in tumor cells and increased attenuation in normal cells.
2. Materials and Methods
2.1. Cell Culture and Viruses
Human colon cancer cell lines LoVo (CCL-229TM) and HCT 116 (CCL-247TM), human lung cancer cell line A549 (CCL-185TM), human hepatocarcinoma cell line Hep G2 (HB 8065TM), human glioblastoma cancer cell line U-87 MG (HTB-14TM), human gastric carcinoma cell line KATO III (HTB-103TM), human pancreatic cancer cell line MIA PaCa-2 (CRL-1420TM), human ovarian cancer cell line SK-OV-3 (HTB-77TM), human bladder cancer cell line UM-UC-3 (CRL-1749TM) and human osteosarcoma cell line 143B, a Thymidine Kinase (TK)-deficient cell line, (CRL-8304TM) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Human esophagus cancer cell line OE19 (n°96071721) was obtained from European Collection of Cell Culture (ECACC). Human head and neck cancer cell line CAL33 was kindly provided by Dr. G. Milano (Centre Antoine-Lacassagne, Nice, France). All cell lines were grown in recommended media supplemented with 10% fetal calf serum (FCS). Fresh human hepatocytes were purchased from Biopredic International (Rennes, France) and maintained in the recommended hepatocyte medium provided by the supplier (Biopredic International).
Primary chicken embryo fibroblasts (CEF) were used for recombination, production and titration of viral vectors. CEF cells were prepared from chicken embryos obtained from fertilized eggs (Charles River SPAFAS) previously incubated 11 or 12 days at 37 °C in a humid atmosphere. Chicken embryos were dissected and treated with a 2.5% (w/v) solution of trypsin. CEF cells were maintained in Eagle-based Medium (MBE) supplemented with 5% fetal calf serum. Wild type vaccinia viruses Wyeth strain (WY, VR-1536TM) and Western Reserve strain (WR, VR-119TM) were obtained from ATCC. Wild type vaccinia virus Copenhagen strain (COP) used in the work described here comes from the Institut Mérieux (Marcy l’Etoile, France). MVA expressing the eGFP gene under the control of the p11k7.5 promoter (MVA-GFP) was constructed and characterized previously .
2.2. 3D Skin Model
The Phenion full-thickness (FT) skin model is a 3D tissue construct that simulates histological and physiological properties of human skin (surface 1.3 cm2). It was purchased from Henkel AG & Company KGaA (Düsseldorf, Germany). According to the supplier’s instructions, tissues were kept at 37 °C under 5% CO2 overnight and fresh, pre-warmed medium was added. Each Phenion FT skin model was infected with 1 × 105 PFU of virus. Cultures were incubated for 7 days at 37 °C and medium was changed every 2 days. After 7 days of infection, medium and skins were frozen. Viral replication was quantified on CEF by plaque assay after sonication.
2.3. Directed Evolution for Selection of Chimeric Vaccinia Virus deVV5
Four viruses COP, WR, Wy and MVA were mixed and used to infect human LoVo cells (1.5 × 106 cells/well in a 6 well-plate) and constituted the passage 1 (LP1). The mix used for the first passage consisted of 1.5 × 104 PFU of COP, WY and WR corresponding to a Multiplicity of Infection (MOI) of 10–2 for each virus and 1.5 × 105 PFU of MVA-GFP corresponding to a MOI 10–1. LP1 was then used in its entirety to infect T-75 tissue culture flask (T75). Supernatants from the second passage were then used in a 10-fold dilution series to infect confluent T75 plates of LoVo cells. Amplification and selection of viral progeny was done by 9 successive passages on LoVo cells by supernatant dilution. The infected T75 were observed for the first signs of cytopathic effect (CPE). To harvest the most potent viruses, cell culture supernatant was harvested from the flask infected with the most concentrated innocula in the 10-fold dilution series that did not show any sign of potent CPE. LP2 and LP3 were harvested after 24 h of infection. Next passages (LP4 to LP9) were harvested at 72 h post infection. LP9 served as crude lysate for a new round of selection on MIA PaCa-2 cells. Twelve passages were performed on MIA PaCa-2 cells as described above. Viral selection and amplification of the strongest chimera was undertaken by decimal dilutions.
2.4. Generation of deVV5-fcu1
deVV5-fcu1 was generated by insertion of the FCU1 gene into the deVV5 TK locus. For TK inactivation, successful recombination events were selected as described . Briefly, CEF were infected with deVV5 at a MOI of 10–2 and incubated at 37 °C for 2 h, then transfected by electroporation using nucleofectorTM 2b device (Amaxa GmbH, Cologne, Germany) with 2 µg of a shuttle plasmid containing the FCU1 gene under the control of the synthetic p11k7.5 promoter and surrounded by the flanking sequence of the vaccinia virus TK gene. The cells were then incubated for 48 h at 37 °C. Double recombination occurred between TK homologous regions in the shuttle plasmid and the wild-type virus, resulting in the insertion of the FCU1 gene into the TK locus of deVV5. Recombinant TK-deleted virus deVV5-fcu1 was selected after infection of the TK-deficient 143B cells in selection medium containing 5-bromo-2-deoxyuridine at final concentration of 150 mg/mL (Sigma, St Louis, MO, USA). Positive TK-plaques were submitted to additional plaque purification cycles in the same conditions. Insertion of the FCU1 sequence into the TK locus was confirmed by multiple PCRs and DNA sequencing.
2.5. In Vitro Cytotoxicity Assay
The lytic capacity was measured using the trypan blue exclusion method. Human tumor cells were transduced in suspension by respective chimeric viruses at the indicated MOI. A total of 3 × 105 cells/well were plated in 6-well culture dishes in 2 mL of medium supplemented with 10% FCS. Cells were then cultured at 37 °C for 5 days and the viable cells were counted by trypan blue exclusion using a Vi-Cell Cell Counter (Beckman Coulter, Brea, CA, USA). All samples were analyzed in triplicate. Mock-infected cells served as negative control and established the 100% survival point for the given assay.
2.6. In Vitro Virus Yield Assay
Growing Hep G2, A549 and OE19 tumor cells were seeded onto 6-well plates at 3 × 105 cells/well. After 24 h in DMEM with 10% FCS, the cells were infected with individual virus at MOI of 10–5 and cells were incubated in fresh growth medium supplemented with 10% FCS until harvesting. Supernatants and cells collected 72 h post infection were submitted to a quick freeze-thaw cycle and sonication to release intracellular viral particles and viral progeny were quantified on CEF by plaque assay. All samples were analyzed in triplicate. To evaluate viral replication human primary cells, human primary hepatocytes were infected in 6-well plates (1 × 106 cells/well) by COP, deVV5 and deVV5-fcu1 at a MOI of 10–4 (100 PFU/well). Cells were incubated in fresh growth medium supplemented with 10% FCS until harvesting. At 48 h post infection, supernatant and cells were collected, freeze-thawed and sonicated and viral progeny was quantified on CEF by plaque assay. To evaluate viral replication on human skin tissue, human reconstituted Phenion skins were infected. The Phenion full-thickness (FT) skin model, a three-dimensional tissue construct that simulates histological and physiological properties of human skin, was purchased from Henkel AG&Co. KGaA (Düsseldorf, Germany). This organotypic epithelial raft culture model was maintained in tissue culture medium according to the manufacturer’s instructions. Each Phenion FT skin model was infected with 1 × 105 PFU of COP, deVV5-fcu1 and deVV5 by infusion (viruses were added directly into the medium). Cultures were incubated for 7 days at 37 °C and medium was changed twice a week. Viral replication both in medium and reconstituted skin was quantified on CEF by plaque assay after 2 cycles of sonication in PBS.
2.7. In Vitro Cell Sensitivity to 5-FC
Human HCT 116 tumor cells in suspension were infected by deVV5 and deVV5-fcu1 viruses at an MOI of 10–5. A total of 3 × 105 cells/well were plated in 6-well culture dishes in 2 mL of medium supplemented with 10% FCS. After 48 h of infection, cells were exposed to various concentrations of 5-FC ranging from 10–7 to 10–3 M. Three days later, cell viability was determined by trypan blue exclusion using a Vi-Cell cell counter. Results are expressed as percentage of viable cells, 100% corresponding to infected cells without 5-FC.
2.8. Cytosine Deaminase Enzymatic Assay
Cytosine deaminase (CDase) activity was quantified by measuring the amount of 5-FU released in the culture media. HCT 116 cells were infected with the different vectors at a MOI of 10–5 and plated in 6-well culture dish (1 × 106 cells/well). After 48 h, 0.1 mM 5-FC was added to the culture medium. Every day for 4 days, 5-FC and 5-FU concentrations in the media were measured by HPLC. Fifty µL of media were quenched with 50 µL of acetonitrile. The samples were vortexed and centrifuged. The organic supernatant was evaporated to dryness and reconstituted in 50 µL of water and analyzed by HPLC using a mobile phase of 50 mM phosphoric acid adjusted to pH 2.1. Results are expressed as the percentage of 5-FU relative to the total amount of 5FC + 5FU after various incubation times with 5-FC.
2.9. DNA Sequencing
Samples from viral DNA were purified with AMPure XP (Beckman Coulter Inc., Brea, CA, USA) beads kit to remove residual cellular DNA and sent for sequencing to the GenomEast platform (IGBMC Microarray and Sequencing platform, Illkirch-Graffenstaden, France). Data from Illumina HiSeq4000 (Illumina Inc., San Diego, CA, USA) in 2 × 100bp paired-end runs were quality trimmed using a Phred score threshold of Q30. Contigs were de novo assembled with SPAdes v3.11.1  following authors instructions and scaffolding was performed with custom script to generate the longest consensus sequence for each viral genome. BLAST 2.7.1  pairwise alignments were used to locate homologous regions between deVV5 and each of the parental strains. Global pairwise alignments were performed using MAFFT v7.017  to highlight the longest regions with strict identity between deVV5 and the corresponding parental genome. Results were reported using Circos v0.69-3  and Geneious v8.1.9, www.geneious.com software .
2.10. Statistical Analyses
Two-tailed Student’s was used to determine significance between treatment groups. Differences were considered significant at p < 0.05.
3.1. Oncolytic Activity of the Four VACV Parental Strains
The oncolytic activity of the four parental VACV strains on various human cell lines was measured to serve as a reference (Table 1). The percentage of surviving cells was calculated 5 days post infection at a MOI of 10–5 (1 infectious viral particle for 105 cells), with 100% of cell survival corresponding to the mock-infected cells. At this MOI, COP was the strongest oncolytic strain tested, therefore it was used as the reference for oncolytic potency throughout this study. As expected and as previously demonstrated , MVA showed no cytotoxicity on tumor cell lines. Table 1 also shows that the tumor cell lines LoVo and MIA PaCa-2 are among the most resistant to all three OVs: WY, WR and COP.
3.2. VACV Shuffling: deVV5 Is a Chimeric Virus with Enhanced Oncolytic Potency In Vitro
A directed evolution strategy employed to generate chimeric VACV comprised two steps (Figure 1). First, a library of viruses was generated by co-infecting LoVo cells with the four viral strains including COP, WY, WR and MVA. Second, amplification of viral progeny under stringent conditions towards clonal isolation of virus candidates was performed by nine successive passages on LoVo cells (LP1–LP9) followed by 12 successive passages on MIA PaCa-2 cells (MP1–MP12), both known to be rather resistant to OV compared to other tumor cell lines (see also Table 1).
During the whole process of chimeric virus selection, several passages were performed by dilution imposing stringent selective pressure towards the genetic selection of a rare recombination event. Oncolytic activity of different passages (LP6 and LP9) were then tested in comparison to the parental COP (Figure 2a). At passage 6 (LP6), no improvement in cytopathic activity was observed except at MOI 10–4 in LoVo cells. At passage 9 (LP9), a clear gain of oncolytic potency was recorded on LoVo cells and a slight gain was observed in OE19 and U-87 MG cells. In contrast, no improvement of oncolytic activity was observed in SK-OV-3, HCT 116, Hep G2 and MIA PaCa-2 cells. Considering these results, LP9 was chosen for another cycle of genetic selection on MIA PaCa-2 which was the least permissive cell line. This second round of selection was performed in order to select an improved oncolytic virus in a wide range of tumor types. Selection by dilution was performed under the same conditions, as described above and in the Materials and Methods section. During the selection process in the MIA PaCa-2 cell line, three passages were tested for their lytic potential. MP6, MP10 and MP12 were compared at low MOI, with the parental COP (Figure 2b). MP12 was found to be more effective than all others. Based on these results MP12 was selected to proceed to clone isolation. Eighteen individual plaque-purified viruses were isolated and screened for their oncolytic potential on the MIA PaCa-2 tumor cell line. The oncolytic potency of individual plaque clones was compared to that of the parental COP and the pool MP12 (Figure 2c). The clones were found heterogenic in potency compared to MP12 pool, suggesting that numerous viruses were present in the pool. About half of the clones seemed to have a similar activity as MP12 (clones 1, 5, 6, 7, 9, 12, 15, 16 and 18) and the other half demonstrated an oncolytic activity close to that observed with COP. The most potent clone at both MOI 10–5 and 10–4 was clone C5. With this clone and using a MOI of 10–5, we have obtained a superior oncolytic activity compared to COP used at MOI 10–4. The clone purity was assessed by evaluation of 20 plaque-purified clones originated from C5, all of which displayed the same phenotype in different tumor cells (data not shown). This clone, termed deVV5, was chosen for further characterizations.
3.3. Genome Analysis
DNA from deVV5 and four parental virus strains was purified and sequenced by Next Generation Sequencing. The use of paired-end short reads and whole genome de novo assembly led to the generation of viral genomes with the core region and one copy of the Inverted Terminal Repeats (ITR) domain. These single-ITR versions of the genomes result from the use of unique k-mers in the contigs assembly process, but the presence of both ITR regions in every viral genome was confirmed by the mean depth coverage which was higher than the one from the core region, with a 2-fold factor (data not shown). Single-ITR versions of the genomes were retained for the sake of clarity in the subsequent paragraph. The resulting genome sequences, MVA (163,668 bp), COP (175,766 bp), WR (181,350 bp), WY (182,933 bp) and deVV5 (172,732 bp) are shown in Figure 3a around the circos plot. The links between the genomes reflect identical DNA sequences longer than 1000 bp between the deVV5 sequence and each of the four parental genomes. Some regions of deVV5 seem to be conserved in several parental strains but long regions (including ITRs) appeared to be specific to only one parental genome. Nevertheless, almost all the deVV5 genome was found to be 100% identical in at least one parental genome. To further investigate the composition of the deVV5 genome, global pairwise alignments were performed to identify the longest identical DNA regions between deVV5 and any parental genome (Figure 3b).
Starting at the 5′ end of the core region, the first 30 kilobases of deVV5 are almost identical to WY sequence, followed by MVA (42 kb). The middle of the core region appears to be a patchwork of regions around 5 kb derived from the four strains. The 3′ end of the core domain is mainly composed of sequences identical to COP (56 kb). Finally, as described above, the 17-kb ITR domain is identical to WY. These observations confirmed that the shuffling resulted in an assembly of three main regions, derived from WY, MVA and COP for the core domain, surrounded by ITR regions derived from WY.
3.4. Arming of deVV5 Leads to Increased Potency
deVV5 was armed with a therapeutic gene, FCU1 , inserted in the TK locus under the control of the strong p11k7.5 VACV promoter, to generate the deVV5-fcu1 variant. The suicide gene FCU1 encodes a protein that catalyzes the conversion of the non-toxic prodrug 5-FC into 5-FU and its derivatives toxic metabolites. Expression of a functional FCU1 protein by deVV5-fcu1 was confirmed by quantification of 5-FU released into the supernatant of infected cells. The analysis of HCT 116 cells supernatants by HPLC showed a progressive release of 5-FU into the extracellular medium of HCT 116 cells infected with the indicated viruses at a MOI of 10–5 and incubated with 0.1 mM 5-FC (Figure 4a). Five days after infection, approximately 60% of 5-FC was converted into 5-FU into the supernatant of deVV5-fcu1 infected cells. To compare the antitumoral activity of deVV5-fcu1 vector alone or combined with 5-FC treatment, the HCT 116 human tumor cell line highly sensitive to 5-FU (data not shown) was infected at a MOI of 10–5 and 5-FC was added to the culture supernatants at a range of concentrations (10–3 to 10–7 M). Cell viability was determined five days post infection by trypan blue exclusion (Figure 4b). The addition of 5-FC had no impact on the viability of mock or deVV5 infected tumor cells. In contrast, the 5-FC conferred increased cytotoxicity in a prodrug dose-dependent manner to deVV5-fcu1 infected human tumor cells. The combination of a low amount of deVV5-fcu1 with 1 mM of 5-FC induced 60% mortality of HCT 116 cells. These results indicate that recombinant deVV5-fcu1, in the presence of the 5-FC prodrug, had acquired an enhanced in vitro anti-tumor activity as compared to deVV5 alone. Together, these in vitro enzymatic activity results demonstrate that deVV5-fcu1 can express a functional therapeutic FCU1 gene and is an efficient vector for viral directed enzyme prodrug therapy (VDEPT) in vitro.
3.5. Increased Oncolytic and Replicative Efficiency of deVV5 and deVV5-fcu1
To determine whether the armed and wild type evolved viruses can replicate and kill cancer cells, a screen of tumor cells from various origins was undertaken (Figure 5a). At low MOI, all tested cancer cells were more susceptible to the chimeric virus deVV5. Numerous cancer cell lines (UM-UC-3, HCT 116, A549 and SK-OV-3) were sensitive to deVV5 infection while remaining resistant to oncolysis by COP. These results were correlated with a significant increase in deVV5 replication in all tumor cell lines tested (Figure 5b).
Compared to parental COP, deVV5 produced five times more viral particles in Hep G2 cells, 30 times more in A549 cells and 90 times more in OE19 cells. The deletion of the TK gene in deVV5 had a slight impact on the virus capacity to kill cancer cells. In most of the tested cell lines, deVV5-fcu1 demonstrated similar lytic activity (Figure 5a) and equivalent replication efficiency (Figure 5b) than the parental deVV5. These results confirmed the oncolytic benefit obtained through the process of directed evolution resulting in genomic blending and the possibility to delete genes without major impact on lytic and replication features of the virus in tumor cells.
3.6. Replication of deVV5 and deVV5-fcu1 on Human Primary Cells
Human hepatocytes and human reconstituted skin (Phenion full-thickness skin model) were used as primary healthy cells to evaluate the in vitro growth profiles of the newly generated viruses. As compared to COP, the chimeric wild type deVV5 demonstrated a reduced replication on primary cells (Figure 5b). A 2-fold and 10-fold reduction of replication were observed respectively in Phenion full-thickness skin model and hepatocytes. Moreover, TK deletion had a clear benefit on deVV5-fcu1 allowing a supplementary 10-fold reduction of replication on human skin and hepatocytes. These results confirmed, and are consistent with previously reports demonstrating, the benefit of deleting TK gene for reducing virus replication in non-cancerous cells . As a consequence, the ratio of viral progeny produced on Hep G2 tumor cells to that in human primary hepatocytes, was largely improved for the deVV5-fcu1 where the transgene was inserted into the TK locus (Figure 5c). Taken together, these results demonstrate the benefit of the recombinant deVV5.
Directed evolution of therapeutic strains of oncolytic viruses is a powerful approach to isolate novel variants with desirable properties. We were able to isolate a new chimeric oncolytic virus, deVV5, by shuffling four VACV genomes, followed by selection under stringent conditions, allowing enrichment of candidates with increased oncolytic potency in vitro. deVV5 displays significant improved oncolytic potency in vitro compared to COP which had shown the highest oncolytic activity of the parental strains. To limit off-target replication and to evaluate its capacity to carry and express foreign therapeutic genes, deVV5 was modified by insertion of the FCU1 gene into the TK locus. Both wild type chimeric virus deVV5 and the TK-deleted-armed deVV5-fcu1 did replicate in most tumor cell lines tested. At very low MOI, i.e., in the range of 10–5, the new variants were able to infect and kill human cancer cell lines that are usually resistant to COP at the same MOI, such as HCT 116 (colon), MIA PaCa-2 (pancreas), SK-OV-3 (ovarian), UM-UC-3 (bladder) and A549 (lung).
The ratio of viral replication in tumor cells compared to that in primary cells, exceeded 400 for deVV5 (Figure 5c), and was further improved 6-fold after TK deletion in deVV5-fcu1. This ratio was improved both by increase of the replicative activity on tumor cell lines and by decrease of the replication on primary cells. Considering the selection process on tumor cells, such a reduction of viral replication in non-cancerous cells was not expected. It is not clear yet whether this might result from components of MVA which is the only non-replicative strain within the four parental viruses. Nevertheless, the increased attenuation in both human skin and hepatocyte cells combined with improved oncolytic potency in vitro suggests that the directed evolution approach is a potent tool to generate improved OVs for cancer treatment.
In this study, we also demonstrated that deVV5 can be armed with a therapeutic transgene without jeopardizing the oncolytic potency and the safety. We and others already demonstrated the potential of the FCU1/5-FC enzyme-prodrug system using a variety of delivery systems including replication-defective viruses [25,28] and replication-selective oncolytic viruses [12,34,35,36,37,38,39]. Our studies with the novel deVV5 confirm these findings.
The sequencing of the new deVV5 revealed the presence of sequences from each of the four parental strains. The presence of each parental genome, at different proportions, highlights the high frequency of homologous recombination between VACV strains. In this work, we decided to report the longest regions of deVV5 with strict identity to parental genomes, likely expected to be the result of homologous recombination events. However, it is important to notice that this representation does not include some shorter regions also identical to other parental strains because VACV genomes share high similarity (at least 90% sequence identity). The three main regions are identical to WY, COP and MVA strains respectively, whereas DNA of WR strain was found at a lower level in the deVV5 genome. The presence of a large region of DNA of the non-propagative MVA, indicates that recombination events appeared quickly, and that chimeric viruses are formed at early stages of the directed evolution process. Further characterization of deVV5 transcriptome and proteome, coupled with comparative genomics approaches should provide more insights on the gain of function associated with the oncolytic phenotype of deVV5.
Prior to testing this virus in the clinical setting, further pre-clinical studies in vivo will be necessary to confirm an advantageous safety profile and improved oncolytic potency. In addition, it will be essential to investigate in vivo the immune profile induced by deVV5 in the tumor microenvironment.
In conclusion, our studies show that directed evolution by shuffling different vaccinia viruses can generate recombinant poxviruses which demonstrate superior oncolytic potency in tumor cells accompanied by increased attenuation in normal cells in vitro. This is a powerful approach and could be adapted to generate novel oncolytic viruses for the treatment of specific cancer indications.
Conception and design: M.R., J.F., D.A. and P.E.; Development of methodology and performance of experiments: M.R., J.F., D.A., A.F., J.K., P.C., A.G., B.G. and P.E.; Analysis and interpretation of data: M.R., J.F., D.A., A.G., B.G. and P.E.; Writing the manuscript: M.R., J.F., B.G., M.L., E.Q. and P.E.
During this study, M. Ricordel was recipient of an Industrial Training Convention for Research (CIFRE, ANR) doctoral fellowship. We thank Catherine Brua for preparation of primary chicken embryo fibroblasts.
Conflicts of Interest
The authors declare no conflict of interest.
- Heinrich, B.; Klein, J.; Delic, M.; Goepfert, K.; Engel, V.; Geberzahn, L.; Lusky, M.; Erbs, P.; Preville, X.; Moehler, M. Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: Studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes. OncoTargets Ther. 2017, 10, 2389–2401. [Google Scholar] [CrossRef] [PubMed]
- Fend, L.; Yamazaki, T.; Remy, C.; Fahrner, C.; Gantzer, M.; Nourtier, V.; Préville, X.; Quemeneur, E.; Kepp, O.; Adam, J.; et al. Immune Checkpoint Blockade, Immunogenic Chemotherapy or IFN-α Blockade Boost the Local and Abscopal Effects of Oncolytic Virotherapy. Cancer Res. 2017, 77, 4146–4157. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Lichty, B.D.; Breitbach, C.J.; Stojdl, D.F.; Bell, J.C. Going viral with cancer immunotherapy. Nat. Rev. Cancer 2014, 14, 559–567. [Google Scholar] [CrossRef] [PubMed]
- Andtbacka, R.H.; Kaufman, H.L.; Collichio, F.; Amatruda, T.; Senzer, N.; Chesney, J.; Delman, K.A.; Spitler, L.E.; Puzanov, I.; Agarwala, S.S.; et al. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J. Clin. Oncol. 2015, 33, 2780–2788. [Google Scholar] [CrossRef] [PubMed]
- Filley, A.C.; Dey, M. Immune System, Friend or Foe of Oncolytic Virotherapy? Front. Oncol. 2017, 7, 106. [Google Scholar] [CrossRef] [PubMed]
- Ribas, A.; Dummer, R.; Puzanov, I.; VanderWalde, A.; Andtbacka, R.H.; Michielin, O.; Olszanski, A.J.; Malvehy, J.; Cebon, J.; Fernandez, E.; et al. Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy. Cell 2017, 170, 1109–1119.e10. [Google Scholar] [CrossRef] [PubMed]
- De Graaf, J.F.; de Vor, L.; Fouchier, R.A.; van den Hoogen, B.G. Armed oncolytic viruses: A kick-start for anti-tumor immunity. Cytokine Growth Factor Rev. 2018. [Google Scholar] [CrossRef] [PubMed]
- Haddad, D. Genetically Engineered Vaccinia Viruses as Agents for Cancer Treatment, Imaging, and Transgene Delivery. Front. Oncol. 2017, 7, 96. [Google Scholar] [CrossRef] [PubMed]
- McFadden, G. Poxvirus tropism. Nat. Rev. Microbiol. 2005, 3, 201–213. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Heo, J.; Reid, T.; Ruo, L.; Breitbach, C.J.; Rose, S.; Bloomston, M.; Cho, M.; Lim, H.Y.; Chung, H.C.; Kim, C.W.; et al. Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nat. Med. 2013, 19, 329–336. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Zeh, H.J.; Downs-Canner, S.; McCart, J.A.; Guo, Z.S.; Rao, U.N.; Ramalingam, L.; Thorne, S.H.; Jones, H.L.; Kalinski, P.; Wieckowski, E.; et al. First-in-man study of western reserve strain oncolytic vaccinia virus: Safety, systemic spread, and antitumor activity. Mol. Ther. 2015, 23, 202–214. [Google Scholar] [CrossRef] [PubMed]
- Foloppe, J.; Kintz, J.; Futin, N.; Findeli, A.; Cordier, P.; Schlesinger, Y.; Hoffmann, C.; Tosch, C.; Balloul, J.M.; Erbs, P. Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus. Gene Ther. 2008, 15, 1361–1371. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Mell, L.K.; Brumund, K.T.; Daniels, G.A.; Advani, S.J.; Zakeri, K.; Wright, M.E.; Onyeama, S.J.; Weisman, R.A.; Sanghvi, P.R.; Martin, P.J.; et al. Phase I Trial of Intravenous Oncolytic Vaccinia Virus (GL-ONC1) with Cisplatin and Radiotherapy in Patients with Locoregionally Advanced Head and Neck Carcinoma. Clin. Cancer Res. 2017, 23, 5696–5702. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Yoo, S.Y.; Jeong, S.N.; Kang, D.H.; Heo, J. Evolutionary cancer-favoring engineered vaccinia virus for metastatic hepatocellular carcinoma. Oncotarget 2017, 8, 71489–71499. [Google Scholar] [CrossRef] [PubMed]
- Hruby, D.E. Vaccinia virus vectors: New strategies for producing recombinant vaccines. Clin. Microbiol. Rev. 1990, 3, 153–170. [Google Scholar] [CrossRef] [PubMed]
- Fenner, F. Genetic studies with mammalian poxviruses. II. Recombination between two strains of vaccinia virus in single HeLa cells. Virology 1959, 8, 499–507. [Google Scholar] [CrossRef]
- Woodroofe, G.M.; Fenner, F. Genetic studies with mammalian poxviruses. IV. Hybridization between several different poxviruses. Virology 1960, 12, 272–282. [Google Scholar] [CrossRef]
- Paszkowski, P.; Noyce, R.S.; Evans, D.H. Live-Cell Imaging of Vaccinia Virus Recombination. PLoS Pathog. 2016, 12, e1005824. [Google Scholar] [CrossRef] [PubMed]
- Koerber, J.T.; Maheshri, N.; Kaspar, B.K.; Schaffer, D.V. Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles. Nat. Protoc. 2006, 1, 701–706. [Google Scholar] [CrossRef] [PubMed]
- Kuhn, I.; Harden, P.; Bauzon, M.; Chartier, C.; Nye, J.; Thorne, S.; Reid, T.; Ni, S.; Lieber, A.; Fisher, K.; et al. Directed evolution generates a novel oncolytic virus for the treatment of colon cancer. PLoS ONE 2008, 3, e2409. [Google Scholar] [CrossRef] [PubMed]
- Kuhn, I.; Bauzon, M.; Green, N.; Seymour, L.; Fisher, K.; Hermiston, T. OvAd1, a Novel, Potent, and Selective Chimeric Oncolytic Virus Developed for Ovarian Cancer by 3D-Directed Evolution. Mol. Ther. Oncolytics 2016, 4, 55–66. [Google Scholar] [CrossRef] [PubMed]
- O’Leary, M.P.; Choi, A.H.; Kim, S.I.; Chaurasiya, S.; Lu, J.; Park, A.K.; Woo, Y.; Warner, S.G.; Fong, Y.; Chen, N.G. Novel oncolytic chimeric orthopoxvirus causes regression of pancreatic cancer xenografts and exhibits abscopal effect at a single low dose. J. Transl. Med. 2018, 16, 110. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Sánchez-Sampedro, L.; Perdiguero, B.; Mejías-Pérez, E.; García-Arriaza, J.; Di Pilato, M.; Esteban, M. The evolution of poxvirus vaccines. Viruses 2015, 7, 1726–1803. [Google Scholar] [CrossRef] [PubMed]
- Mayr, A.; Stickl, H.; Müller, H.K.; Danner, K.; Singer, H. The smallpox vaccination strain MVA: Marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism. Zentralbl. Bakteriol. B 1978, 167, 375–390. [Google Scholar] [PubMed]
- Erbs, P.; Findeli, A.; Kintz, J.; Cordier, P.; Hoffmann, C.; Geist, M.; Balloul, J.M. Modified vaccinia virus Ankara as a vector for suicide gene therapy. Cancer Gene Ther. 2008, 15, 18–28. [Google Scholar] [CrossRef] [PubMed]
- Husseini, F.; Delord, J.P.; Fournel-Federico, C.; Guitton, J.; Erbs, P.; Homerin, M.; Halluard, C.; Jemming, C.; Orange, C.; Limacher, J.M.; et al. Vectorized gene therapy of liver tumors: Proof-of-concept of TG4023 (MVA-FCU1) in combination with flucytosine. Ann. Oncol. 2017, 28, 169–174. [Google Scholar] [CrossRef] [PubMed]
- Quoix, E.; Lena, H.; Losonczy, G.; Forget, F.; Chouaid, C.; Papai, Z.; Gervais, R.; Ottensmeier, C.; Szczesna, A.; Kazarnowicz, A.; et al. TG4010 immunotherapy and first-line chemotherapy for advanced non-small-cell lung cancer (TIME): Results from the phase 2b part of a randomised, double-blind, placebo-controlled, phase 2b/3 trial. Lancet Oncol. 2016, 17, 212–223. [Google Scholar] [CrossRef]
- Erbs, P.; Regulier, E.; Kintz, J.; Leroy, P.; Poitevin, Y.; Exinger, F.; Jund, R.; Mehtali, M. In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. Cancer Res. 2000, 60, 3813–3822. [Google Scholar] [PubMed]
- Nurk, S.; Bankevich, A.; Antipov, D.; Gurevich, A.A.; Korobeynikov, A.; Lapidus, A.; Prjibelski, A.D.; Pyshkin, A.; Sirotkin, A.; Sirotkin, Y.; et al. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J. Comput. Biol. 2013, 20, 714–737. [Google Scholar] [CrossRef] [PubMed]
- Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
- Katoh, K.; Misawa, K.; Kuma, K.; Miyata, T. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002, 30, 3059–3066. [Google Scholar] [CrossRef] [PubMed]
- Krzywinski, M.; Schein, J.; Birol, I.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S.J.; Marra, M.A. Circos: An information aesthetic for comparative genomics. Genome Res. 2009, 19, 1639–1645. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Ricordel, M.; Foloppe, J.; Pichon, C.; Sfrontato, N.; Antoine, D.; Tosch, C.; Cochin, S.; Cordier, P.; Quemeneur, E.; Camus-Bouclainville, C.; et al. Cowpox Virus: A New and Armed Oncolytic Poxvirus. Mol. Ther. Oncolytics 2017, 7, 1–11. [Google Scholar] [CrossRef] [PubMed]
- Simpson, G.R.; Horvath, A.; Annels, N.E.; Pencavel, T.; Metcalf, S.; Seth, R.; Peschard, P.; Price, T.; Coffin, R.S.; Mostafid, H.; et al. Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic HSV as an intravesical therapy for superficial bladder cancer. Br. J. Cancer 2012, 106, 496–507. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Dias, J.D.; Liikanen, I.; Guse, K.; Foloppe, J.; Sloniecka, M.; Diaconu, I.; Rantanen, V.; Eriksson, M.; Hakkarainen, T.; Lusky, M.; et al. Targeted chemotherapy for head and neck cancer with a chimeric oncolytic adenovirus coding for bifunctional suicide protein FCU1. Clin. Cancer Res. 2010, 16, 2540–2549. [Google Scholar] [CrossRef] [PubMed]
- Quirin, C.; Rohmer, S.; Fernández-Ulibarri, I.; Behr, M.; Hesse, A.; Engelhardt, S.; Erbs, P.; Enk, A.H.; Nettelbeck, D.M. Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy. Hum. Gene Ther. 2011, 22, 389–404. [Google Scholar] [CrossRef] [PubMed]
- Kaufmann, J.K.; Bossow, S.; Grossardt, C.; Sawall, S.; Kupsch, J.; Erbs, P.; Hassel, J.C.; von Kalle, C.; Enk, A.H.; Nettelbeck, D.M.; et al. Chemovirotherapy of malignant melanoma with a targeted and armed oncolytic measles virus. J. Investig. Dermatol. 2013, 133, 1034–1042. [Google Scholar] [CrossRef] [PubMed]
- Hammer, K.; Kazcorowski, A.; Liu, L.; Behr, M.; Schemmer, P.; Herr, I.; Nettelbeck, D.M. Engineered adenoviruses combine enhanced oncolysis with improved virus production by mesenchymal stromal carrier cells. Int. J. Cancer 2015, 137, 978–990. [Google Scholar] [CrossRef] [PubMed][Green Version]
Figure 1. The directed evolution process. Representation of the different steps of the directed evolution process used to select a new Poxvirus chimera with improved oncolytic activity.
Figure 2. Oncolytic activities during the different steps of the directed evolution process. (a) Percentage of surviving cells 5 days after infection with passage 6 (LP6), passage 9 (LP9) and Copenhagen stain (COP). Human tumor cells were infected at a MOI of 10–5 and 10–4 with COP and the indicated passages and cell viability was determined by trypan blue exclusion. The parental COP was used as reference. The results are presented as a mean of triplicate experiments ± SD. (b) Percentage of surviving cells after infection of MIA PaCa-2 tumor cell line with the indicated passages at MOI 10–5 and 10–4. Cell viability was determined 5 days after infection. The parental COP was used as reference. The results are presented as a mean of triplicate experiments ± SD. (c) Percentage of surviving cells 5 days after infection of MIA PaCa-2 tumor cell line with the indicated clones at MOI 10–4 and 10–5. The parental COP and MP12 were used as references. The results are presented as a mean of triplicate experiments ± SD.
Figure 3. Homology maps between deVV5 and each parental genome. (a) De novo sequenced viral genomes are shown around the circos plot (COP, orange; MVA, red; WY, blue; WR, green; deVV5, white). Only one ITR is displayed (black). Blast results of deVV5 regions greater than 1 kb with 100% identity are linked by color-coded ribbons to the corresponding hit. (b) deVV5 genome annotated with results from global pairwise alignments. Each annotation corresponds to the longest region with 100% identity to the corresponding parental genome. deVV5 core and single ITR region are also displayed.
Figure 4. Functionality and efficacy of FCU1 expressed by deVV5. (a) Conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and release of 5-FU in the cell culture supernatant. HCT 116 cells were infected with the indicated vector at a MOI of 10–5 and then incubated with 0.1 mM 5-FC from day 2 to day 5 post infection. The relative concentration of 5-FC and 5-FU in the culture supernatant was measured by HPLC. The results are expressed as the percentage of 5-FU released relative to the total amount of 5-FC + 5-FU. Each data point represents the mean of triplicate determinations ± SD. (b) In vitro sensitivities of infected human tumor cells to 5-FC. HCT 116 human tumor cells were mock-infected or infected with deVV5 and deVV5-fcu1 at a MOI of 10–5. After 48 h, cells were treated by increasing concentrations of 5-FC. Cell survival was determined 3 days later as described in Materials and Methods section. Cell viability results are expressed as the percentage of viable cells in the presence and absence of the prodrug. Values are represented as mean ± SD of triplicate determinations.
Figure 5. Both chimeric and recombinant chimera are able to infect and kill cancer cells leaving primary cells intact. (a) Oncolytic activities of COP, deVV5 and deVV5-fcu1 by measuring the cell viability 5 days after infection of different cancer cell lines. Tumor cells were infected with the indicated viruses and cell viability was determined as described in the Materials and Methods section. The MOI used for each cell line was adjusted according to its susceptibility to COP infection. The MOI used for the infection was MOI 10–5 (3 PFU/well) for UM-UC-3, SK-OV-3, CAL33, A549 and Hep G2 cells, MOI 10–4 (30 PFU/well) for OE19, MIA PaCa-2 and HCT 116 cells and MOI 10–3 (300 PFU/well) for KATO III cells. (b) Replication in tumor cell lines and in primary human cells. Tumor cells were infected at MOI 10–5 and harvested 3 days post infection. Human primary hepatocytes were infected with 100 PFU/well and harvested 3 days post infection. 3D Phenion FT skin models were infected with 1.105 PFU and harvested 7 days post infection. Viral progeny production was determined by plaque titration. Results are expressed as viral fold amplification (corresponding to output/input ratio). Asterisks denote statistical significance compared to COP (p < 0.05). (c) Ratio between viral production obtained in Hep G2 hepatocarcinoma cells and hepatocytes for the three viruses: COP, deVV5 and deVV5-fcu1.
Table 1. Percentage of surviving cells infected by four strains of vaccinia virus (VACV) used in this study. Various human tumor cell lines were infected by the four parental VACV strains (MVA, WY, WR and COP) at Multiplicity of Infection (MOI) 10–5. Survival was determined 5 days later, as described in the Materials and Methods section. Cell viability results are expressed as the percentage of viable cells relative to non-infected cells. Values are expressed as mean ± SD of three individual infections.
|A549||94.5 ± 3.6||90.2 ± 3.2||58.6 ± 3.7||47.8 ± 4.8|
|LoVo||98.6 ± 2.7||90.4 ± 5.9||70.8 ± 6.2||73.1 ± 5.2|
|MIA PaCa-2||103.2 ± 4.2||92.8 ± 2.9||89.4 ± 2.4||78.1 ± 1.5|
|U-87 MG||102.8 ± 4.8||64.6 ± 1.6||41.0 ± 0.8||43.6 ± 0.3|
|Hep G2||97.3 ± 3.9||66.8 ± 4.9||70.9 ± 9.4||13.6 ± 2.7|
|HCT 116||99.2 ± 5.4||91.5 ± 3.4||94.6 ± 3.4||55.1 ± 7.5|
|OE19||105.2 ± 6.2||78.9 ± 2.2||77.6 ± 3.3||70.3 ± 0.7|
|SK-OV-3||95.4 ± 7.3||59.8 ± 1.8||99.7 ± 1.5||42.9 ± 1.3|
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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People campaigning to be allowed to swim in Bristol’s Floating Harbour have been warned they will be prosecuted and could be fined £1,000 if they continue to stage illegal ‘protest swims’.
That was the message from the deputy harbour master, who told around 25 people ‘wild swimming’ in the Cumberland Basin this morning that it was dangerous and not allowed.
It was the second ‘swim protest’ this week, after campaigners led by local businessman Johnny Palmer stepped into the water to bring attention to their campaign.
They say the water quality, which had been poor for years, is now good enough for Bristol City Council to consider the idea of allowing wild swimming in the Basin, or at other specific locations in the Floating Harbour, under controlled conditions.
The campaigners point to places like Copenhagen, where inner city bodies of water, canals and rivers have special swimming areas.
But the council today reinforced its policy of not allowing swimming of any kind - even by experienced wild swimmers under controlled conditions.
The council said that the Floating Harbour is a working docks, the water quality isn’t good enough, and swimming there under any circumstances is not safe. Council chiefs are also worried that the campaign might encourage other people less experienced to get in the water - and there have been a number of deaths over recent years in the harbour.
There are signs around the docks, including at the Cumberland Basin, that make it clear that no swimming is allowed and anyone in the water could be fined £1,000.
On Bank Holiday Monday, Bristol Live reported how a group of around a dozen wild swimmers got into the water of the Cumberland Basin, went for a swim and were then ejected from the water by the Harbour authorities.
On Friday morning, numbers had swollen to between 25 and 30, and this time everyone had exited the water before the Deputy Harbour Master arrived on foot, accompanied by a Harbour Master patrol boat.
The Deputy Harbour Master told the swimmers that what they were doing was dangerous, unsafe and illegal - and they would be prosecuted.
“To everyone here, I’d just like to point out - this is illegal what you’re doing,” he announced.
“Whether it changes in the future… but I ask you to stop, politely, and prosecutions will be made from now on,” he said.
The deputy harbour master returned to specifically warn the last two remaining swimmers who were still getting changed again, to tell them they would be prosecuted if they swam again.
“I’ll give you a warning that if I see you here again, it will result in a civil prosecution by the council,” he told them.
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Mr Palmer told Bristol Live he had emailed the Harbour Master’s office asking for a meeting to discuss the ban on swimming in the Floating Harbour.
In an open letter to the Harbour Master after Friday morning’s incident, Mr Palmer again said he wanted to meet to discuss the possibility of allowing open water swimming.
“We are highly disappointed that despite repeated contact you have not accepted my invitation to meet and discuss the issue of swimming in the harbour,” Mr Palmer said.
“In this meeting we can discuss how our group can use our voice and experience to help promote harbour safety – as well as make your job easier.
“I truly believe that working together we can make the harbour overall a much safer place for all.
“Please note that at this stage we are looking to work with you to achieve positive outcomes for all stakeholders. The raised-voice and authoritative approach that some representatives of your office are using is not conducive with this aim.
“The current ‘prohibition’ approach to swimming right now is not working and there is a better way. History shows what prohibition results in,” he added.
Yoga teacher and outdoor swimming coach Amelia Paul, from Swimoga, was one of those who took part in the illegal swim.
“I’m a very keen wild swimmer. At the moment I swim in Clevedon, I swim in the River Avon on the other side of Bristol - I have to get in my car, I have to drive, and I live less than half a mile from Bristol harbour,” she said.
“I’ve visited places like Copenhagen, and I really feel it is a great time to get conversations going, as a lot of people here do, about how safely we can all use the harbour, at specific times, and obviously when the water quality is good, as well, so that we can get in the water and use the water for swimming.
“I would love to help people who have never wild swum before, who live in the city, to be able to come and get in an open body of water which is on their doorstep, rather than ask them to have to drive places, or get on the bus and go to places outside of the city centre,” she added.
“I think it’s a really good time - the rise in outdoor swimming has gone up by 300 per cent since lockdown. The outdoor swimming society are saying how popular it’s become.
“I feel like it is a really good time to start asking the question - not should we be doing it, but how can we do this safely? And I’m really excited about getting this conversation going,” she said.
But Bristol City Council and the Harbour Master maintain swimming there is dangerous and no-one should be doing it.
In a statement issued on Monday by the council, and reaffirmed on Friday, they outlined the reasons why swimming is banned.
“Bristol’s harbour is a working area in an integral part of the city and wild swimming in this area is dangerous and against the local by-laws designed to ensure safety," a spokesperson said.
“The health and safety of all harbour users is our first priority and, along with other members of the Bristol Water Safety Partnership, our harbour staff work hard to maintain a safe environment along the waterway and prevent accidents and so that people can enjoy the range of attractions on offer.
“As the Harbour Authority, the council conducts regular safety checks and has ensured measures such as life rings, improving visibility of quayside ladders and installing more life chains in certain high-risk areas are in place.
“As with any large harbour or waterway, we cannot completely remove all risks and would urge people not to endanger themselves and others by swimming in this area," he added
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text:
List of scoring systems .
During austere conditions when there is a large demand on healthcare services and the resources are limited for different reasons, there should be a special way of managing patients and victims in order to make the most benefit to the community. Trial of first come, first served will lead to losing most of the seriously injured patients because they will reach late if they reached a healthcare facility. In addition, day-to-day work protocols with full resources also are not the optimum to offer for the whole community during a major incident. Triage has been created and evolved in military medical services to face mass casualty with limited resources and then transferred to civilian life to deal with mass casualty incidents. Applying triage to patients created some interference with medical bioethics if those applied on individual bases, but if applied in the whole picture of state or country, we can understand its rations.
- military triage
- major incident
- emergency department triage
God created human beings and honored them over other creatures; therefore, keeping life is one of utmost urges. This urge to save lives is challenged in time of major incidents when patients’ needs are exceeding care resources. Moreover, with the increase in global population and escalation in the costs of healthcare, more patients are visiting emergency departments (EDs) all over the world to cut expenses and bypass remote appointments. Most EDs today adopt a triage system to prioritize patients who need urgent care.
2.1 Linguistic definition
“The process of determining the most important people or things from amongst a large number that require attention” .
2.2 In medical use
It is the sorting of victims by giving them grade to prioritize them for treatment and transportation in order to maximize the number of survivors in major incidents and war victims . According to the assigned grade, patients will have their priority in attending by healthcare givers, investigations, and operation rooms.
The term triage is similar to “rationing” and “allocation” which is practiced on a daily basis in every field. For the term triage to be applied for a situation, there are three prerequisites that must be fulfilled:
There is shortage of resources in comparison to the needs.
There should be a system set to triage by the health body or facility.
Trained health personnel should do the triage .
3. History of triage
Triage started as war time medical effort driven by the increased number of wounded and shortage of resources. In addition the need for manpower during wars affected the priorities in triage in some armies.
It is believed that the first time triage used in military medicine to prioritize treatment for the wounded was by Baron Dominque Jean Larry (8 July 1766–25 July 1842). He made rules that the wounded are treated by the severity of their clinical conditions regardless of the rank; even enemies were treated in the same way .
The next milestone in triage was attributed to British rear admiral John Crawford Wilson (1834–4 July 1885) . He differentiated between the severity levels of the wounded; he wrote in his book Outlines of Naval Surgery: “If a case should be hopeless, or the man apparently dying, an operation then would be useless” . He was sorting wounded soldiers into three categories: slight, serious, and fatal. This was the base for further division in triage system by creation of the expectant zone. In the triage system created by Dominique, all serious cases are treated in the same level.
Triage in the American civil war was depending on the first-come, first-served basis regardless of the severity, salvageability, or best use of limited resources .
The World War I with the development of more lethal weapons like machine guns and chemical gases with a large number of wounds that could be treated pushed the military surgeons to apply and refine triage protocols. This has led to the concept of “The greatest good of the greatest number.” This is the rule for triage practiced in military and civilian life during major incidents now . This rule means that at time of limited resources and facing huge demand, some patients can be saved if long time and large amount of resources devote to them, but this will not be done. The reason to not offering help to those patients is that we can save much more number of wounded patients (who are less critical) using the same resources during the same time. We may save 10 patients instead of one. The pressure of escalating numbers of wounded soldiers with limited fighters in the battles made some health strategic planners in the armies to give higher priorities to patients that can be treated and sent back to front war lines rapidly over seriously injured patients that need urgent intervention for long duration. Winslow listed the two objectives of triage as “1st, conservation of manpower; 2nd, the conservation of the interest of the sick and wounded” .
In the World War II, the weapons were more developed with the introduction of tanks and air forces. On the other hand, medications and health services improved. The health strategic planners still concentrate on supporting the troops. They direct resources for soldiers who are able to fight rather than injured or diseased ones.
With the improvement of transportation and less dependence on the manpower in modern wars, it is rarely nowadays needed to leave somebody without treatment for the sake of others. The triage decision now is to which facility best transfer the patient and what is the optimum method of transportation.
4. Moral and ethical issues in triage
Health system ethics has been developing and improving since the eighteenth century by the First Geneva Conventions (1859) and Nuremberg Act . In 1979, Beauchamp and Childress published their book Principles of Biomedical Ethics . They put four main principles which are:
Respect of autonomy
4.1 Requirements for a triage system
To have a triage system, there are three requirements to be fulfilled:
There should be shortage of resources in comparison to the need.
There should be a system set by health authority to be used in such circumstances (point A).
There are personnel trained on the system who will implement it.
If there is no shortage, then no need to use triage, but for every patient, the health facility will do its best to treat the patient.
A triage system should be set by the health authority or facility administration to be followed by anyone doing this task. The aim of this step is to look for the benefit of the community and the population as a whole and not to just part of it.
Trained personnel to practice the triage to ensure the justice and prevent personal preference.
During major incidents, there are situations in which the triage officer should take some decisions that may be hard and not in the best interest of some patients. The decisions made by the planners in the First and Second World Wars and before are made not by patients’ will or his best interest and benefit. Below is the discussion of the principles one by one:
Respect of autonomy: During a normal life, this is the first patient’s right. No intervention should be done unless the patient understands the issue fully and accepts it. For this reason, the informed consent is needed to be signed by the patient. In time of major incidents with a large number of patients present, there should be prioritization of patients according to system agreed upon by the hospital or health authority. In time of major incidents, absolute knowledge of the whole community needs is predominant over individual liberty. It is sure that some people will not be happy with delaying them regardless of their presentation time or their degree of severity.
Beneficence: In 1964, the World Medical Association (WMA) developed the Declaration of Helsinki as a set of ethical principles for experimentation on human beings. The declaration strongly emphasizes (a) that the concern for the interests of the subject must always prevail over the interests of science and society and (b) that ethical considerations must always take precedence over laws and regulations. Those principles cannot be applied during disaster conditions. The overall benefit to the community should overrule the personal benefit, and the concept of “maximum benefit to maximum number” should be used to maximize community benefit and welfare.
Later in the chapter, there is a section regarding exceptions to the general rules.
Non-maleficence: Non-maleficence means doing non-harming or inflicting the least harm possible to reach a beneficial outcome . In this meaning trying to save as much as possible of the community can explain depriving some patients from treatment or delay them until suitable time and resources are available. In this issue we may not consider all people as the same, for example, if there is a healthcare giver and a fighter that are wounded, then we should not count each as one person, because when the healthcare giver is treated, he will help in saving the other.
Justice: In justice we mean that each patient should take what he needs and no one should be disadvantaged or deprived from treatment. People may misunderstand the meaning well and have high expectations to treat all patients as the highest-priority patient. To explain this we should differentiate between equality and justice. The first one means that everybody should receive the same amount. For example, a patient with fracture can wait for days, while the unconscious patient with multiple injuries needs rapid assessment and a full management plan rapidly implemented. This is justice; each patient will take time and resources according to the severity of the condition.
Ownership of resources is challenged in time of major incidents, and the hospital should accept and treat any patient involved in the incident (according to the plan) .
5. Special circumstances in triage
Although triage depends mainly on patients’ injury severity, there are conditions which oblige the officer to modify his triage decision or in austere condition to decide to whom priority of care is given. The triage officer should look to the whole picture of the community, putting in his mind the aims at that particular time he is doing it and the full resources in addition to the type of patients he is dealing with. The following are examples of special conditions which need special care and by no means are they exhaustive:
Children: Dealing with children is sensitive not only from the emotional side but also the practical side. Children have more expected life span than old people, and in time of limited resources with the equality of other factors, priority should be given to children for the sake of the community.
Pregnant women: In dealing with pregnant women, we are dealing with two lives; therefore, they have double importance and should take priority.
Emergency services personnel: All those personnel should not be counted as one person; if we give them priority and save them, they will help in saving more lives. We give them the value of the expected number of lives they may save. In addition taking care of someone injured rapidly will encourage other to put all their efforts, knowing that their colleagues will treat them in high priority if they are injured, and this will improve the quality of care given to all patients.
People with special skills or knowledge or with special importance: There are some people who possess some special knowledge and skills or have some special importance to the country. Those should also get special treatment and priority. This will need confirmation of their status and priority from local or national authority to recognize them during major incidents.
The surrounding circumstance: If there is a critical need to manpower like in war condition, for example, then the triage officer may make the highest priority to simple cases that can be treated with minimal resources and time and go back to combat area. Another example is facing floods and waiting for central help, until extra help reaches them, and there is a desperate need for all hands even the slightly wounded; otherwise, the whole area and local community will have grave outcomes.
Combination of the abovementioned conditions needs the triaging officer to put his priority at that single moment.
6. Common triage systems
We can divide triage systems into several categories:
Military triage system
Major incidents in civilian life
Emergency department triage system which is used for managing patients on a daily basis
Military triage system: The military triage differs from civilian life because in many occasions there is chaos and many of the infrastructures are not present or destroyed by the combat. Another reason is that the troops are usually located outside the cities where there are no or small services and they need to build their own treatment and evacuation system. The healthcare in present time is provided to every wounded for two reasons. First, there is no dependence on manpower like previous battles, and, second, there is huge improvement in communication and transportation tools and equipment. Now every wounded soldier is treated, but the difference is in time and place.
Major incidents in civilian life: Civilian triage started around 200 years after the military one. The first triage system is simple triage and rapid treatment (START) which is a method used in the field to rapidly sort and prioritize patients during major incidents according to the severity of their injuries. It was developed in 1983 in California [12, 13]. Later there will be discussion of other systems that are developed later.
The triage systems used in military and major incidents “that occur in civilian life” are the same, and it will be discussed in combination. They differ in the infrastructures supporting each one, with clear overlap between them.
Table 1 shows some of scoring systems used to evaluate the severity of the injuries which is the base for triage.
|1970||AIS||Abbreviated injury scale|
|1974||GCS||Glasgow Coma Scale|
|1974||ISS||Injury severity score|
|1980||TRISS||Trauma injury and severity score|
|1981||APACHE||Acute physiological and chronic health evaluation|
|1987||PT||Pediatric trauma score|
|1987||OIS||Organ injury scale (AAST)|
|1988||PRISM||Pediatric risk of mortality score|
|1989||RTS||Revised trauma score|
|1989||T-RTS||Triage version of RTS|
|1990||ASCOT||A severity characterization of trauma|
|1994||UST||Uniform scoring system for trauma (Utstein style)|
|1994||APSC||Acute physiology score for children|
|1996||ICD-9-CM||ICD-9 clinical modification based on AIS and ISS|
|1996||TOXALSTM||Toxic advanced life support TM|
|2001||ASPTS||Age-specific pediatric trauma score|
|2002||PAAT||Pediatric age-adjusted TRISS|
|2003||START||Simple triage and rapid treatment|
|2003||JUMP-START||Pediatric version of START|
There are different categories for triage in major incidents. They are the physiological and the anatomical methods.
The physiological systems are easily learned and need simple training; any health personnel can be trained and perform it. Moreover it can be reproduced easily and is a reliable method of following up the patient’s condition. On the other hand, it is time-consuming and not suitable for incidents with a huge number of victims.
Anatomical systems of triage are fast and depend on visual recognition of injuries. These methods need a good amount of experience in injuries and when the patient needs surgery. It is difficult to reproduce the results as it is subjective and not objective. A very large number of victims is suitable for this type of triage.
After knowledge of the anatomical and physiological condition of patients, the triage officer needs to know the comorbidities to and other circumstances (discussed above in the section of special situations) to give the patient the final triage level.
The most common triage systems used in major incidents are as follows:
Glasgow Coma Scale: It is a scoring system used to evaluate the patients with coma or disturbed consciousness (Table 2). It was first described by Graham Teasdale and Bryan Jennett in 1974 and was used as a practical method to evaluate patients with brain injury and a good method to communicate the patient’s condition between different healthcare providers or facilities. The findings use the scale guide initial decision-making and monitor trends in responsiveness that are important in signaling the need for new actions . Each point will be given a number: the maximum score is (15) and means the patient is fully conscious, and the lowest is (3) which is clearly seriously injured. The reactions to be noticed are:
There are studies comparing the full GCS to the motor reaction alone. It is believed that motor response is better predictive of patients’ condition than full GCS . Recently the use of GCS in triage is objected because it is time-consuming and can be interpreted in different combinations for the same score .
Simple triage and rapid treatment (START): It is currently widely used in the United States and many other countries. People can be easily trained on it and used to sort victims rapidly into four categories: red, yellow, green, and white (or black). It depends on the ability of the victim to walk and then give the green category. If the patient cannot walk and has any alteration in his level of consciousness or vital signs, then he is categorized as red. If there is no alteration, he will be yellow. If no breathing and unconscious, then he is considered dead (white). Figure 1 shows the flowchart for START triage system .
JumpSTART: It is the pediatric version of the START system; the main difference is the trial with the child to do airway maneuver and short resuscitation trial in the field before declaring death of the child. In addition to considering the change of heart rate in different age groups, Figure 2 shows the flowchart for JumpSTART triage system.
Triage-revised trauma score (T-RTS): This system depends on physiological parameters of the patient at the time of evaluation. It is found to be a good indicator of the severity of the injury . It depends on three physiological parameters which are (1) Glasgow Coma Score (GCS), (2) systolic blood pressure (BP), and (3) respiratory rate (RR). Each factor will be given a score, and then the scores of the three factors are summed: the more the score, the better the condition of the patient and vice versa. Table 3 shows the different items in T-RTS and their interpretation.
There are several other systems, but they are used to assess and predict prognosis in trauma patients and not used for prioritizing them during response to major incidents.
Emergency department triage systems: The emergency department shares with the military life the inability to control the number and time of patients presented. With timeliness of the care and relative scarce of resources, in addition to the increase in the demand on beds and blocked access to other services, more patients are seeking medical care in emergency departments . To make risk assessment of patient introduced to the ED and make the best benefit of the resources, a five-level triage system was introduced in Ipswich, Australia. This system was validated and adopted in several states in Australia and formed the base for different national systems in several countries in the world. The triage is done by experienced nurses, will give patients one of the five levels depending on the urgency of the conditions. Table 4 shows the five levels in emergency department triage and the period allowed before attending the patient .
|To pressure||2||Words||3||Normal flexion||4|
|Categories and urgencies in the emergency triage system|
|Category||Urgency of the condition|
|One||Patients in this category should be attended immediately when presented|
|Two||Patients will have priority and seen in the next doctor available. By passing que of patients present|
|Three||To place the patient’s file at the front of the waiting list|
|Four||Wait for their que or may be advised to go to a primary health facility|
|Five||Discharged from emergency side and advised to visit the primary health facility|
There are several triaging systems in different countries like the Manchester triage system which is widely used in the UK hospitals and the Canadian triage system (CTAS) and others, all using five levels and basically similar in sorting patients. There are minimal differences in the bench mark for the time frame each category should be seen within.
7. Wrong triage
With the rush and chaos occurring during response to major incidents, there are mistakes that may be committed by the triaging officer. Under-triage and over-triage are the wrong decisions that may occur:
Under-triage: It is defined as “A term of art referring to underestimating the urgency of the condition of a person arriving in A&E and not prioritizing his or her management over that of a patient with less urgent needs” . It is a medical problem and may result is serious bad outcomes because it deprives a patient from the resources he/she needs. What is the level of acceptable risk of under-triage? The CDC puts the threshold as 20%, but in practice it is 35% . If the validation depends on the injury severity score (ISS) and any patient with a score of >15 transferred to a hospital without trauma center, then the acceptable level is 5% . It is found that patients with under-triage have less mortality rate than patients with right triage to the trauma centers because they have better GCS score, blood pressure, and base deficit .
Over-triage: A term of art referring to unintentionally overestimating the urgency of the condition of a person arriving in A&E (casualty) and prioritizing that person’s management over that of a patient with more urgent needs . This is a less risky medical mistake. Its effect is on the limited resources during a major incident in terms of human resources, stuff used, or space occupied, e.g., intensive care beds, CT scan, operating theater, etc.; due to its low risk to patients, it is agreed that 25–50% is accepted .
Triage is a key step in managing major incidents properly. It is not contradicting bioethics, but it is looking from a different focus to make the best to the whole community. It has no rigid rules, and the triage officer must look for different aspects of resources and patients’ situation to make the best triage decision leading to most benefits for all.

domain: medical


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Several notable big leaguers have officially hung up the cleats this offseason, or given an indication (sort of) about when they might be retiring. Here’s the latest from some more veterans about when they may or may not consider calling it a career…
- R.A. Dickey is entering the last year of his contract with the Blue Jays and the 41-year-old tells VICE Sports’ John Lott that he’ll sit down with his family after the season to decide on his future. Right now, “we’re all kind of undecided. We just like to stay in the moment,” Dickey said. If he does return, it would be for a situation that suits his family and likely a one-year contract. “I think I will be an attractive option for a lot of teams because I will not be requiring a multi-year deal, and I will probably be asking less than what my market value will be….And for me, it’s not necessarily about the money at this point. It’s about the right fit, and how close to home I’d be, and are there direct flights, and what is the clubhouse policy for kids, all that stuff,” Dickey said. Despite his age, the knuckleballer is as steady an innings-eater as any in baseball, averaging 219 frames per season since 2011. Given the lack of starting pitching available in next winter’s free agent market, Dickey is probably correct in assuming he would get a lot of interest.
- Speaking of ageless right-handers, Bartolo Colon tells Kristie Ackert of the New York Daily News that plans to pitch as long as he’s healthy. “I take it year by year. We’ll see what happens for me,” Colon said. The 42-year-old fan favorite is preparing for his 19th season in the bigs, returning to the Mets rotation until Zack Wheeler makes his midseason return from Tommy John rehab.
- Left-hander Cesar Jimenez hasn’t arrived at the Brewers’ Spring Training camp, and while the team hasn’t heard anything official yet, Jimenez might be retiring, Tom Haudricourt of the Milwaukee Journal Sentinel tweets. Jimenez appeared in 16 games with the Crew last season after he was selected off waivers from Philadelphia in August, and he signed a minor league deal to return to Milwaukee in January. Jimenez, 31, has a 4.05 ERA over 104 1/3 career innings and parts of six big league seasons with the Brewers, Phillies and Mariners.
No Soup For Yu!
In other words, Dickey will not be signing with the White Sox.
No Soup For Yu!
I posted a comment that (as of this posting) is awaiting moderation. I hope it’s not because R.A.’s last name contains a naughty word.
Bartolo Colon is just a gift from nature. How does he pull it off?
Seems like it would be worthwile to point out what city or state it is that Dickey calls home if you’re going to quote the part about him probably wanting to play close to or a direct flight from home.
Exactly what I was thinking
I agree too. Too funny.
“Despite his age, the knuckleballer is as steady an innings-eater as any in baseball, averaging 219 frames per season since 2011”
From 2011 – 2015, among *ALL* pitchers in baseball, Dickey ranks at #4 with with 1097 innings pitched. The only guys that are ahead of Dickey are J. Shields (1135), C. Kershaw (1128), F. Hernandez (1107.2)
It’s not simply a ‘steady innings-eater’.; it’s ELITE production in that category.
I would submit that “… as any in baseball …” covers that.
His ‘termination’ from the National post was really disheartening to read. Good for John Lott for finding a new gig elsewhere.
Jays should trade Dickey to the Mets for Syndergaard and d’Arnaud. No, that’s a crazy deal, nobody would ever do that deal. Wait …
Don’t forget outfielder w. buccera (spelling) too. Young up and coming five tool player. Will likely have to be added to 40 man next off season and maybe looking at 2017 September call up. He could be even better than trav and Noah. Now that would end up going down as one of the best trades in franchise history and a top trade in mlb history if things play out at the current pace.

domain: sports


IEA Report Urges Global Alignment on Hydrogen Emission Intensity for a Cohesive Framework
The International Energy Agency (IEA) has released a new report titled “Towards Hydrogen Definitions Based on their Emissions Intensity,” aimed at informing policymakers, hydrogen producers, investors, and researchers ahead of the G7 Climate, Energy and Environmental Ministerial meeting in April 2023.
This report expands on the IEA’s “Net Zero by 2050: A Roadmap for the Global Energy Sector” and is part of a series addressing sector-specific details of the roadmap.
The report highlights the importance of creating a cohesive, global understanding of hydrogen emissions intensity. This understanding is crucial for informing policymakers, hydrogen producers, investors, and researchers, as they navigate the transition to clean energy sources.
The report assesses the greenhouse gas (GHG) emissions intensity of different hydrogen production routes, aiming to establish a standardized emissions accounting framework. This shift away from the current use of color-based terminology or other impractical terms is expected to provide transparency and facilitate international hydrogen supply chain development.
In this context, the report emphasizes the need for a globally agreed-upon framework for emissions accounting. By adopting such a framework, market fragmentation can be reduced and interoperability can be increased, ultimately enabling more effective investments in international hydrogen supply chains.
The report builds on the IEA’s “Net Zero by 2050: A Roadmap for the Global Energy Sector” and is part of a series of reports prepared for the G7 focusing on the sectoral details of the roadmap. With this new report, the IEA aims to provide valuable insights and guidance for the development of regulation and certification schemes related to hydrogen emissions intensity.


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It was that kind of week…
Welcome to Balls of Steel’s AFL Beat!
Round 20 Recaps and Highlights
Strap yourselves in, kids, because this week’s games gave us quite a ride. It all started on Friday morning when I woke up early enough to catch the second part of the third quarter of the Richmond Tigers-Geelong Cats match.
By then, the Tigers had run out to a healthy 24 point lead and I seriously thought about turning the game off. But something told me to keep watching and the Cats mounted a tremendous fourth quarter comeback that ended this way:
Oh, if only Gary Ablett’s shot had gone in! Here are the full game highlights:
On proper Friday night, the first game featured the Essendon Bombers fighting for their finals lives against the Hawthorn Hawks. It was an awesome game to watch and it too came down to the last two minutes:
The whole game was crazy and back and forth and the Bombers just ran out of time. Highlights:
So, have you had enough thrilling finishes? Nope, not yet! The North Melbourne Kangaroos had led the Brisbane Lions only for the Lions to fight back then led again and the Lions would come back. This pattern held up until the fourth. And then this happened:
Jesus, Brisbane! You have a free kick 25 yards at a 45 degree angle with less than a minute left, you MILK THAT CLOCK AND TAKE A DROP PUNT EVERY DAY OF THE WEEK!
Talk about pulling a loss out of a win. Here are the highlights:
The next game was The Showdown between the Adelaide Crows and the Port Adelaide Power. The Crows desperately needed this game to keep their super slim finals hopes alive.
Predictably, it went down to the wire:
See? That’s how you milk a clock! Highlights:
You would think we would be done with awesome finishes, but you’d be wrong. Sydney and Collingwood played a thriller that also came down to the end:
How did he score that goal? I mean that’s unbelievable! He pulled the win out of a hat! Amazing! Here are the full game highlights:
As if to give us a break from the heart-stopping finishes, the St. Kilda Saints were kind enough to give up their lead over the Western Bulldogs midway through the second quarter. The Doggies thanked them and never looked back in a 103-68 (so close!) win. Here are the highlights:
Come North American Saturday night, the Footy Gods decided we’d had enough and all three games went as expected.
In the first game, the GWS Giants took care of the Carlton Blues by 105 points (151-46). The remarkable part about this match was that the Giants suffered injuries and had to play with 18, 17, and even 16 men during the fourth quarter.
AND THEY STILL OUTSCORED THE BLUES IN THAT QUARTER!
The next match, between the Melbourne Demons and the Gold Coast Suns, also had blowout written all over it and the Demons did not disappoint. They won by 96 and here are the highlights:
Lastly, the Perth Derby between West Coast Eagles and the Fremantle Dockers did not figure to be a blowout, but methinks the Dockers are already looking up all-inclusive packages to Bali. The final score was 142-84 and here are the highlights:
Let’s take a look at the ladder, shall we?
|2||West Coast Eagles||14||5||0||56||124.1|
|7||Port Adelaide Power||12||7||0||48||114.9|
|10||North Melbourne Kangaroos||11||8||0||44||110.1|
|15||St. Kilda Saints||4||14||1||18||75.1|
|17||Gold Coast Suns||4||15||0||16||62.2|
Here is the Balls (TM) graph:
Crazy how one loss means so much, huh? Things are getting REALLY interesting now…
Footy Tipping Contest
Here is where we are after Round 20:
|P||TIPPER||Round Tips||Round Margin||TOTAL Tips||TOTAL Margin|
|9||Joliet Jake Delhomme||3||3||96||602|
|10||Game Time Decision||5||21||95||653|
A big week for me sees me vault over Spanky and Litre into 3rd place! Meanwhile, Game Time Decision continues his climb up the ladder with a place inside the top ten within reach.
For those of you not putting the tips in, this is for you:
DON’T FORGET TO PUT THE FOOTY TIPS IN!!!
AFL Round 21
Here is the schedule for this week (all times Pacific):
Friday, August 10, 2018
2:50 AM (Late Thursday Night) – Essendon Bombers v St. Kilda Saints at Marvel Stadium
It’s nice that I don’t have to wake up extra early to watch this game. I assume the Bombers will be PISSED about last week’s close loss to the Hawks and will take it out on the Saints.
8:45 PM – Hawthorn Hawks v Geelong Cats at the MCG
This is a YUUUGE game for the Cats. In essence, it’s a must-win as the Roos are tied on points and have an opportunity to build percentage against the Doggies.
9:10 PM – Gold Coast Suns v Richmond Tigers at Metricon Stadium
The only way the Tigers lose this is if the team plane gets lost on its way to Queensland and the VFL team has to play the Suns.
11:35 PM – Port Adelaide Power v West Coast Eagles at Adelaide Stadium
It should be a great game as Port tries desperately to salvage the season and not fall out of the Eight. West Coast is sitting pretty in second and only a collapse will force them out of Top Two.
Saturday, August 12, 2018
2:25 AM (Late Friday Night) – Collingwood Magpies v Brisbane Lions at Marvel Stadium
This is another game where a team with a close loss last week will look to make up for it against an inferior opponent. Although, the Lions have shown fight and really should have won last week…
2:25 AM (Late Friday Night) – GWS Giants v Adelaide Crows at UNSW Canberra Oval
This is probably the Match of the Round. It’s the Crows’ last-ditch effort to stay in the race. Will the injuries cause problems yet again for the Giants?
8:10 PM – North Melbourne Kangaroos v Western Bulldogs at Marvel Stadium
The Roos have a nice and easy schedule at the end. They must take advantage of it and beat the Doggies this week.
10:20 PM – Melbourne Demons v Sydney Swans at the MCG
This could be the Match of the Round Part Two. Sydney has played well on the road and will want to prove they can win at the MCG.
11:40 PM – Fremantle Dockers v Carlton Blues at Optus Stadium
Fremantle has not shown anything in the last few weeks. The Blues have been embarrasing to watch. Something has to give!
That’s all for this week! As you can see, the outcomes of individual games greatly affect the ladder as the competition is super tight. I’m hoping we get more games that go to the final two minutes.
That way, I can play more Iron Maiden. See you next week!

domain: hobby


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Goldeneye on Bond pop-up sound
This is an issue I've raised on the DStv Now forum before. Not sure if it's related.
Sound on Goldeneye (broadcast) seemed to have issues with the 5.1. The centre sound was incredibly low, with the rear and surrounds a lot louder. So in scenes you were hearing the effects and some partial music, but actual gunshots and dialogue were almost silent.
Did anyone else notice this? I've set another recording for this weekend, when the film repeats. It wasn't watchable with those sound issues.

domain: entertainment


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Welcome to CLIP - the Crew List Index Project
CLIP was set up to improve access to the records of British merchant
seafarers of the late 19th century and has gathered the largest
database of entries from crew lists. This site gives information about:
NB! CLIP is confined to merchant seafarers on British
registered ships for the years 1861 to 1913.
This site provides guidance and finding aids to help
you carry out your own research. Sorry, we are not able to carry out privately commissioned research or provide transcripts or copies of crew lists.
The CLIP crew name index is available online at

domain: library