scholarly journals Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1007
Author(s):  
Jiří Gregor ◽  
Kateřina Radilová ◽  
Jiří Brynda ◽  
Jindřich Fanfrlík ◽  
Jan Konvalinka ◽  
...  
Keyword(s):  
Thermodynamic Analysis ◽  
Influenza A ◽  
Polymerase Activity ◽  
Second Phase ◽  
Missense Mutations ◽  
Wild Type ◽  
Resistance Development ◽  
Third Phase ◽  
Major Mutation ◽  
Control And Prevention

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2020 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Genetic Background ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Resistant Virus ◽  
Wild Type ◽  
Fitness Advantage

AbstractFavipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2021 ◽  
Vol 17 (6) ◽  
pp. e1008937
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Wild Type Virus ◽  
Resistant Virus ◽  
Wild Type ◽  
Drug Pressure ◽  
Fitness Advantage

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.

scholarly journals PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1046
Author(s):  
Ahmed Mostafa ◽  
Sara H. Mahmoud ◽  
Mahmoud Shehata ◽  
Christin Müller ◽  
Ahmed Kandeil ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Influenza A Virus ◽  
Influenza A ◽  
Polymerase Activity ◽  
Wild Type ◽  
Genetic Compatibility ◽  
Bronchial Epithelial ◽  
Replication Efficiency ◽  
Avian Influenza A Virus ◽  
Replication Fitness

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1EGY and H9N2EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2EGY, expressed individually and in combination in the genetic background of H5N1EGY, were genetically compatible with the other H5N1EGY segments. At 37 °C the replication efficiencies of H5N1EGY reassortants expressing the H9N2EGY polymerase subunits PB2 and PA (H5N1PB2-H9N2EGY, H5N1PA-H9N2EGY) were higher than the wild-type H5N1EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1PB2-H9N2EGY, but reduced for H5N1PA-H9N2EGY. At 33 °C and 39 °C, H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY replicated to higher levels than the wild-type H5N1EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1EGY. Genetic analysis of the polymerase-encoding segments revealed that the PAH9N2EGY and PB2H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1EGY enabled the mutant virus H5N1PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

PENERAPAN MODEL QUANTUM LEARNING UNTUK MENINGKATKAN HASIL BELAJAR AUTOCAD SISWA KELAS X TEKNIK PEMESINAN SMK NEGERI 1 STABAT

2013 ◽  
Vol 5 (1) ◽  
Author(s):  
Abdul Hasan Saragih
Keyword(s):  
Positive Response ◽  
Phase 1 ◽  
First Grade ◽  
Learning Model ◽  
Second Phase ◽  
Phase 2 ◽  
Phase 3 ◽  
The Third ◽  
Third Phase ◽  
Quantum Learning

This classroom research was conducted on the autocad instructions to the first grade of mechinary class of SMK Negeri 1 Stabat aiming at : (1) improving the student’ archievementon autocad instructional to the student of mechinary architecture class of SMK Negeri 1 Stabat, (2) applying Quantum Learning Model to the students of mechinary class of SMK Negeri 1 Stabat, arising the positive response to autocad subject by applying Quantum Learning Model of the students of mechinary class of SMK Negeri 1 Stabat. The result shows that (1) by applying quantum learning model, the students’ achievement improves significantly. The improvement ofthe achievement of the 34 students is very satisfactory; on the first phase, 27 students passed (70.59%), 10 students failed (29.41%). On the second phase 27 students (79.41%) passed and 7 students (20.59%) failed. On the third phase 30 students (88.24%) passed and 4 students (11.76%) failed. The application of quantum learning model in SMK Negeri 1 Stabat proved satisfying. This was visible from the activeness of the students from phase 1 to 3. The activeness average of the students was 74.31% on phase 1,81.35% on phase 2, and 83.63% on phase 3. (3) The application of the quantum learning model on teaching autocad was very positively welcome by the students of mechinary class of SMK Negeri 1 Stabat. On phase 1 the improvement was 81.53% . It improved to 86.15% on phase 3. Therefore, The improvement ofstudent’ response can be categorized good.

Mixed-Methods Evaluation as Scaffold to the Development of a Mobile Health Application to Support the Prescription of Wheelchairs and Postural Support Devices (Preprint)

2020 ◽  
Author(s):  
Adriana Klein ◽  
Roseli de Deus Lopes ◽  
Rodrigo Suigh
Keyword(s):  
Mixed Methods ◽  
Mobile Health ◽  
Second Phase ◽  
The Third ◽  
Evaluation Approach ◽  
Mixed Methods Evaluation ◽  
Usability Tests ◽  
Third Phase ◽  
Postural Support ◽  
Support Devices

BACKGROUND EasySeating is a mobile health (mHealth) app that supports the prescription of wheelchair and postural support devices (WPSD). It can be used by occupational therapists (OT) and physiotherapists (PT) who prescribe WPSD. The app offers a standardization of the prescription procedure, showing images, metrics and details that guide the prescriber to decide on the best equipment. It was developed with an iterative mixed-methods evaluation approach. Objective: The aim of this study was to investigate the processes involved in the prescription of WPSD and to propose, develop and evaluate a mHealth to support OT and PT prescribers. OBJECTIVE The aim of this study was to investigate the processes involved in the prescription of WPSD and to propose, develop and evaluate a mHealth to support OT and PT prescribers. METHODS This study was divided into three phases and was carried out as an iterative process composed of user consulting/testing (using a mixed-methods evaluation approach), system (re)design and software development. The first phase consisted of the collection of qualitative and quantitative data to map and understand the users requirements and of the development of the first prototype (v1) of the app. This data collection was performed through semi-structured interviews with 14 OT and PT prescribers, 5 specialized technicians and 5 WPSD users. The second phase aimed at improving the overall functionality of the app and consisted in the development, test and evaluation of the prototypes v1, v2, v3 and v4. A total of 59 prescribers tested and evaluated these prototypes by means of open interviews, semi-structured questionnaires and focus groups. The third phase focused in the usability aspects of the app. It consisted in the development and test of the prototype v5. Eight technology specialists assessed its usability through heuristics evaluation. RESULTS Data collected in phase one indicated there is a lack of standardization on the prescription of postural support devices (PSD). A divergent nomenclature for the PSDs was also found and classified in eight categories. These information guided the development of the first prototype of the EasySeating app. Phase two results pointed that the prescribers value the insertion of the app into their clinical practice, as it accelerates and increases the quality of the evaluation process and improves the organization of the prescription information. Significant suggestions for the improvement of the app were given during the users tests, including the use of images to represent the PSDs. The usability tests from the third phase revealed two strong issues that must be solved: the need of greater feedback and failures in the persistence of the input data. CONCLUSIONS This study demonstrated that there is a lack of systematization of the WPSD prescription process. The evaluation of the developed EasySeating app demonstrated that there is a potential to standardize, integrate and organize the WPSD prescription information, supporting and facilitating the decision making process of the prescribers. CLINICALTRIAL This study was approved by the Research Ethics Board of the Universidade de São Paulo (registered protocol n°53929516.6.0000.0065) URL - http://plataformabrasil.saude.gov.br/login.jsf

Foucault as Superauthor

2019 ◽  
Vol 29 (2) ◽  
pp. 104-125
Keyword(s):  
Free Speech ◽  
Second Phase ◽  
Contemporary Society ◽  
Sovereign Power ◽  
Ancient Greek ◽  
Main Motivation ◽  
Critical Attitude ◽  
The Third ◽  
Third Phase ◽  
Lecture Courses

Three phases in Foucault’s examination of authorship and free speech were essential to him throughout his life. They can be linked to such texts as the three lectures “What is an Author?” (first phase), “What is Critique?,” and “What is Revolution?” (second phase), and the two lecture courses, “Fearless Speech,” and “The Courage of Truth” (third phase). Initially, Foucault merely describes the founders of discursivity (hence, “superauthors”), among whom he reckoned only Marx and Freud, as the sole alternative to his own conceptualization of the author function, which is exhibited en masse in contemporary society. He then modifies his views on superauthorship by making Kant the paradigm and by linking his own concept of free speech to a Kan-tian critical attitude. However, Foucault claims only the half of Kant’s philosophical legacy that is related to the study of the ontology of the self.The article advances the hypothesis that the sovereign power of speech, which can be found in Marx and Heidegger and in generally in the concept of “superauthorship,” becomes unacceptable for Foucault. During the third phase, the danger of a tyrannical use of free speech compels Foucault to make a number of fruitful but questionable choices in his work. He focuses on a single aspect of free speech in which a speaker is in a weaker position and therefore has to overcome his fear in order to tell the truth. Foucault associates this kind of free speech with the ancient Greek notion of parrhesia, which according to his interpretation means “fearless speech”; however, this reading is not always supported by the ancient Greek sources. Foucault’s deliberations bring him to the radical conclusion that free speech transforms into performative “aesthetics of existence.” Foucault’s main motivation for pursuing this line of thought all through his life was to investigate his own abilities and powers as an author

scholarly journals Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James D. Allen ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Virus Infections ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

scholarly journals Hand Surgery in Thailand

2021 ◽  
Vol 13 (01) ◽  
pp. 35-41
Author(s):  
Sunyarn Niempoog ◽  
Kiat Witoonchart ◽  
Woraphon Jaroenporn
Keyword(s):  
World War Ii ◽  
Traffic Accidents ◽  
Hand Surgery ◽  
Vital Role ◽  
Second Phase ◽  
World War ◽  
Third Phase ◽  
Foreign Countries ◽  
General Surgeons ◽  
Set Up

AbstractModern hand surgery in Thailand started after the end of World War II. It is divided into 4 phases. In the initial phase (1950-1965), the surgery of the hand was mainly performed by general surgeons. In 1965-1975, which was the second phase, many plastic surgeons and orthopaedic surgeons graduated from foreign countries and came back to Thailand. They played a vital role in the treatment of the surgery of the hand and set up hand units in many centers. They also contributed to the establishment of the “Thai Society for Surgery of the Hand,” which still continues to operate. In the third phase (1975-2000), there was a dramatic development of microsurgery because of the rapid economic expansion. There were many replantation, free tissue transfers, and brachial plexus surgeries in traffic and factory-related accidents. The first hand-fellow training program began in 1993. In the fourth phase (since 2000), the number of hand injuries from factory-related accidents began declining. But the injury from traffic accidents had been increasing both in severity and number. Moreover, the diseases of hand that relate to aging and degeneration had been on the rise. Thai hand surgeons have been using several state-of-the-art technologies such as arthroscopic and endoscopic surgery. They are continuing to invent innovations, generating international publications, and frequently being invited as speakers in foreign countries.

scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

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