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Structural characterization of superbug proteins involved in regulating beta-lactam resistanceWilke, Mark Steven 05 1900 (has links)
The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-lactams, (2) expression of PBPs with low-affinity for β-lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance.
The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction.
The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.
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Investigation of exudative epidermitis and ear necrosis in pigsPark, Jeonghwa 23 December 2011 (has links)
This thesis is an investigation of two common skin conditions of pigs: exudative epidermitis (EE) and ear necrosis (EN). The cause of exudative epidermitis and risk factors are well understood, however the study was prompted because of reports of treatment failure. A survey of veterinary practitioners (n=15) and pork producers (n=58) was conducted to determine which treatments are commonly used. Amongst farmer respondents topical treatments were often used and in serious cases injectable penicillin G was administered. Thirty farms with a history of EE were visited and skin samples taken from affected pigs. The antimicrobial resistance pattern for isolates of Staphylococcus hyicus and Staphylococcus aureus revealed that almost all isolates were resistant to penicillin G and ampicillin. In addition, certain isolates of S. hyicus as well as S. aureus were shown to possess the mecA gene which is associated with resistance to methicillin. The presence of widespread resistance to penicillin G among staphylococci isolates suggests a reason for poor treatment response. The presence of the mecA gene in staphylococci other than S. aureus recovered from pigs has not been reported before and is of interest from a public health standpoint.
A second study investigated EN. The causative agent(s) and the associated risk factors are not well understood. Eleven case farms were visited and skin biopsies and oral swabs taken from pigs in early, mid and late stages of the disease. Bacteriological culturing was performed for staphylococci and spirochetes as well as histological examination of the biopsy samples. Farm-level risk factors were assessed on 14 case farms and 9 control farms. Staphylococci were generally recovered in abundance from the majority of samples but spirochetes were not cultured and only identified microscopically in a small number of tissue samples. Histology revealed that the disease appeared to occur first as a lesion on the epidermal surface that caused tissue damage and led to subsequent invasion of the dermis. This pathogenesis was consistent with the hypothesis that staphylococci colonize the skin surface and produce exfoliating toxins. Ear biting was noted to be commonly present and may be an important contributing factor. / Ontario Pork
Animal Health Strategic Initiative Fund
Ontario Ministry of Agriculture, Food and Rural Affairs(OMAFRA)
Ontario Veterinary College, University of Guelph
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Structural characterization of superbug proteins involved in regulating beta-lactam resistanceWilke, Mark Steven 05 1900 (has links)
The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-lactams, (2) expression of PBPs with low-affinity for β-lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance.
The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction.
The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.
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The effect of resuscitation fluids on beta lactam antibiotic pharmacokinetics in interstitial tissue in acute thermal injuryKanchanamala Ranasinghe Unknown Date (has links)
Advantages and disadvantages of administration of resuscitation fluids in burns patients have been discussed at length. However, the effect of resuscitation fluids on tissue physiological endpoints and tissue antibiotic distribution is scarcely reported, yet clinically crucial. The preliminary studies of this thesis involved evaluation of the literature and the development of a non - recovery anaesthetized rat model of burn injury suitable for the study of plasma and tissue physiological changes and antibiotic pharmacokinetics (PK). Therefore, the first series of the studies for this thesis was designed to examine the relative effects of a range of crystalloid and colloid-containing resuscitation fluids on tissue pH following burn injury in a rat model. The secondary aims were to examine the effects of these fluids on tissue blood flow, plasma protein extravasation (PPE) and evaporative water loss (EWL). In these studies we confirmed that the burn injury and fluid resuscitation were accompanied by a tissue acidosis. Administration of Lactated Ringers’ Albumin (LRA) and Lactated Ringers’ Dextran (LRD) effectively attenuated the degree of tissue acidosis in the thermally injured and non injured sites for 180 minutes post burn and the transepidermal water loss (TEWL) on the non injured sites during the first 60 minutes of the acute phase of burn injury. The second phase of the work was designed to assess the changes in antibiotic distribution with the administration of these different fluids in plasma as well as in interstitial tissues in the burn and the non burn sites. This study showed that for cephalothin (4g/kg body weight, administered intravenously (IV)), Lactated Ringers solution (LR) and Hypertonic Saline (HS) showed similar plasma PK with Time > Minimum inhibitory concentration (MIC) (> 180 minutes) in plasma. However, the antibiotic tissue distribution was more skewed towards lower levels for HS when compared with LR. For piperacillin (18g/kg body weight, administered IV), Time > MIC was considerably low comparatively, being only 55 min for both LR and HS. Antibiotic concentrations did not reach the MIC with LRA resuscitation. When considering the interstitial tissues, Time > MIC for cephalothin was lower than HS with LR on both the burn and the non burn sites. T > MIC for piperacillin was zero for all fluids in both burn and non burn sites. The major finding of this study was that with LRA resuscitation, antibiotic distribution was significantly lower than seen with LR and HS for both antibiotics studied in the interstitial tissue fluid space in both the burn and non burn sites. The final phase of the work was designed to study the apparent permeability co efficient of Keratinocytes (KC) to antibiotics in the presence of simulated pH changes observed in burn tissue in thermal injury using colloids and crystalloids. This study found that there was no significant difference between the basolateral and apical concentrations of antibiotics observed neither with the different pH values nor with time. However, there was definitely a significant difference in the apparent permeability of the cells with LR vs LRA and that the permeability was higher with LR than LRA. This study confirmed that the presence of LR allows greater permeation of the antibiotic into the KC, and also that with LRA resuscitation, the antibiotic tends to stay at higher concentrations in the interstitial compartment. These studies demonstrate that choice of resuscitation fluid following burn injury can affect both changes in tissue physiology and antibiotic distribution, warranting further study in both animal models and patient populations.
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Processo intensificado de hidrolise enzimatica de penicilina G e purificação dos produtos em reator multi-estagio e contra-corrente / Intensificated process of hydrolysis of penicillin G and purification of the products in a multi-stage couter-current reactorFerreira, Juliana de Souza 16 July 2004 (has links)
Orientadores: Telma Teixeira Franco, Adrianus Johannes Straanhof, Lucas Antonius Maria van der Wielen / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-05T06:02:03Z (GMT). No. of bitstreams: 1
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Previous issue date: 2004 / Resumo: Este trabalho estuda a hidrólise enzimática da penicilina G (PenG) em ácido 6-aminopenicilânico (6-APA) e ácido fenil acético (PAA). Em um reator contra-corrente multi-estágio e em baixos valores de pH, a reação enzimática ocorre na fase aquosa e os produtos são separados entre a fase aquosa e a fase orgânica (acetato de butila). Além disso, em pH baixo, a cristalização do 6-APA ocorre quando concentrações de PenG são altas. Ambos fenômenos deslocam o equilíbrio no sentido de conversão do substrato, promovendo alta produtividade. A primeira etapa deste trabalho refere-se à avaliação da atividade e estabilidade da penicilina amidase a baixo pH e na presença de acetato de butila (BuAc). A enzima apresentou máxima atividade na faixa de pH 8,0 - 9,0 e permaneceu estável mesmo em pHs baixos (3,0 - 6,0) num período de incubação de até 32 dias. Embora a atividade enzimática sofra um decréscimo de aproximadamente 80%, isto não representa empecilho para sua utilização no emprego da hidrólise de PenG em processo contínuo e bifásico (água e BuAc) em pH baixo. O efeito de PenG, PAA e BuAc na cristalização do 6-APA e os parâmetros cinéticos de cristalização também foram avaliados. Os resultados mostraram que as impurezas não exerceram efeito sobre a cristalização de 6-APA, na faixa de pH entre 4 e 5 e nas concentrações de impurezas de 0,55 mM - 3,0 mM. A avaliação da cinética de cristalização possibilitou o uso de um modelo que pode predizer as taxas de cristalização de 6-APA. Um modelo quantitativo foi desenvolvido para o cálculo do pH e das concentrações do substrato e dos produtos nos estágios do reator contra-corrente. Os dados fornecidos pelo modelo podem ser utilizados para otimizar as condições de operação como: estágio de alimentação, vazão volumétrica das fases e concentração inicial do substrato. Na última etapa deste trabalho foi feita uma revisão bibliográfica sobre biorreatores extrativos em que são apresentadas as vantagens de cada configuração e as restrições dos processos biocatalíticos. Através desta revisão, foi verificado que o uso de um sistema, formado por agitadores acoplados a hidrocic1ones em série, pode representar uma opção adequada de reatores multi-estágio contra-corrente para a hidrólise de PenG em escala de laboratório / Abstract: This work studies the enzymatic hydrolysis of penicillin G (PenG) into 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). In a multi-stage countercurrent reactor and at low pH, the enzymatic reaction takes place in the aqueous phase and the products are separated between the aqueous phase and organic phase (butyl acetate - BuAc). Furthermore, 6-APA crystallization occurs at low pH when PenG concentrations are high. Both phenomena shift the equilibrium towards conversion of substrate, favoring high productivity. The first step of this work, concerns the evaluation of activity and stability of penicillin amidase at low pH and in the presence of butyl acetate (BuAc). The enzyme presented the maximum activity in the pH 8.0 - 9.0 and remained stable at low pHs (3.06.0) during at least 32 days. Although the enzyme activity decreased by 80%, this does not represent a drawback in the application of a biphasic (water and BuAc) and continuous PenG hydrolysis at low pH. The effect of PenG, PAA and BuAc in APA crystallization and the kinetic parameters were also analyzed. The results showed that impurities have no effect on APA crystallization, in the pH range 4 - 5 and in the impurity concentrations of 0.55 mM - 3.0 mM. The evaluation of crystallization kinetics allowed the use of a mo del that predicts the APA crystallization rates. A quantitative model was developed in order to calculate the pH and substrates and products concentrations in the countercurrent reactor. The data provided by the model can be used to optimize the operation conditions: stage of feed, flow rate of phases and initial substrate concentration. In the last step of this work, a literature review concerning extractive bioreactor was made. This review presents the advantages of each configuration and the restrictions of the biocatalytic processes. Through this review, a integrated system of mixers and hydrocydone was suggested as an appropriate option of multi-stage countercurrent reactor for PenG hydrolysis in laboratory scale / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química
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Reclaiming the Activity of Lost TherapeuticsTelussa, Rallya 01 July 2016 (has links)
ESKAPE pathogens are notorious in causing nosocomial infections and escaping current antibiotic treatments. There has been a dramatic increase in nosocomial infections accompanied with a decrease in the number of antibiotics developed, leading to significant increase in morbidity and mortality among patients. In an attempt to combat this problem, derivatives of ciprofloxacin, rifabutin and beta-lactam antibiotics were synthesized and tested against the ESKAPE pathogens. From minimum inhibitory concentration assays, 4 ciprofloxacin analogs and 8 beta-lactam analogs were found to be effective against multiple bacterial species. Additionally, 12 rifabutin analogs and 23 beta-lactam analogs were potent against single bacterial species, primarily toward methicillin-resistant Staphylococcus aureus (MRSA) at a concentration of ≤ 25 µg mL-1. Based on the effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), three rifabutin analogs were selected for further testing. Two rifabutin analogs (DU644 and DU645) were found to possess between a one to twofold mean increase of inhibitory activities, while the other rifabutin analogs (DU650) demonstrated up to a twofold decrease of inhibitory activity when compared to the parent drug. These compounds were then examined for their bactericidal and antibiofilm activities against MRSA. From these assays, we found that DU644 and DU645 were 4 times more bactericidal and antibiofilm against MRSA when compared to the parent drug. In addition, rpoB mutation validation results confirmed that modification of these rifabutin derivatives at the C3 and C4 positions, and bearing an imidazolyl ring carrying substituted spiropiperidyl ring, did not change their mechanism of action towards the beta-subunit of RNA polymerase. Cytotoxicity testing performed using human hepatocellular carcinoma epithelial cells (hepG2) showed that at concentrations ranged from 1.25 µg mL-1 to 25 µg mL-1, DU644 and DU645 showed very low toxicity. Collectively, structural drugs modifications of these obsolete drugs are able to restore their antibacterial activities against MRSA, which is notable as the most infectious nosocomial pathogen. Therefore, further development and application of rifabutin analogs might be beneficial for medical use to combat MRSA infections.
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Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologiesKrallis, Myrsini January 1997 (has links)
The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
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Structural characterization of superbug proteins involved in regulating beta-lactam resistanceWilke, Mark Steven 05 1900 (has links)
The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-lactams, (2) expression of PBPs with low-affinity for β-lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance.
The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction.
The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
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Synthesis of Paclitaxel AnalogsXu, Zhibing 29 November 2010 (has links)
Paclitaxel is one of the most successful anti-cancer drugs, particularly in the treatment of breast cancer and ovarian cancer. For the investigation of the interaction between paclitaxel and MD-2 protein, and development of new antagonists for lipopolysaccharide, several C10 A-nor-paclitaxel analogs have been synthesized and their biological activities have been evaluated. In order to reduce the myelosuppression effect of the paclitaxel, several C3â ² and C4 paclitaxel analogs have been synthesized and their biological evaluation have been studied. / Master of Science
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Studies on an N-terminal nucleophile hydrolase and enzymes of clavulanic acid biosynthesisIqbal, Aman January 2008 (has links)
(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure.
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