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Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin
Get PDFWe recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPlx_N and pSiMPlx_C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41-1 to reconstitute the enzyme conferring resistance towards the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin and puromycin. Here we introduce another pair for use with spectinomycin/streptomycin broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance towards ampicillin, kanamycin, chloramphenicol and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance towards antibiotics.Unfunde
Baseline dasabuvir resistance in Hepatitis C virus from the genotypes 1, 2 and 3 and modeling of the NS5B-dasabuvir complex by the in silico approach
Get PDFBackground: Current combination treatments with direct-acting antiviral agents (DAAs) can cure more than 95% of hepatitis C virus (HCV) infections. However, resistance-associated substitutions (RASs) may emerge and can also be present in treatment-naïve patients. Methods, results and discussion: In this study, a semi-pan-genotypic population sequencing method was developed and used to assess all NS5B amino acid variants between residue positions 310 and 564. Our method successfully sequenced more than 90% of genotype (GT) 1a, 1b, 2b and 3a samples. By using the population sequencing method with a cut-off of 20%, we found the dasabuvir RASs A553V and C445F to be a baseline polymorphism of GT 2b (8 out of 8) and GT 3a (18 out of 18) sequences, respectively. In GT 1a and 1b treatment-naïve subjects (n=25), no high-fold resistance polymorphism/RASs were identified. We further predicted dasabuvir's binding pose with the NS5B polymerase using the in silico methods to elucidate the reasons associated with the resistance of clinically relevant RASs. Dasabuvir was docked at the palm-I site and was found to form hydrogen bonds with the residues S288, I447, Y448, N291 and D318. The RAS positions 316, 414, 448, 553 and 556 were found to constitute the dasabuvir binding pocket.Uppsala-Örebro Regional Research Council, the Erik, Karin and Gösta Selander Foundation and the Scandinavian Society for Antimicrobial Chemotherap
Identification of putative drug targets and annotation of unknown proteins in Tropheryma whipplei
No full textTropheryma whipplei (T. whipplei) is the causative agent of Whipple's disease and blood culture-negative endocarditis. Due to the variability of symptoms, the disease is often poorly diagnosed. Treatment for this bacterial infection is often lengthy, and improper uptake of antibiotics has resulted in relapses in many patients. In the present study, using available bioinformatic tools and databases such as the Cluster Database at High Identity with Tolerance (CD-HIT), the Basic Local Alignment Search Tool for proteins (BLASTp), the Database of Essential Genes (DEG), and the DrugBank database, 13 putative drug targets were identified in T. whipplei by subtractive genome analysis that could be targeted with currently available drugs (experimental or approved). Further, a 3D model was generated for one of these putative drug targets, the T. whipplei DNA ligase, and in silico docking was performed with pyridochromanone and adenosine-derived inhibitors using the AutoDock Vina. Additionally, many of the T. whipplei protein sequences in the National Center for Biotechnology Information (NCBI) protein database were unknown/uncurated. Using available web servers e.g. the KEGG Automatic Annotation Server (KAAS), the BLASTp, the Conserved Domain Architecture Retrieval Tool (CDAT) and the Protein families (Pfam), the function/remote/domain homology for nearly 80% of these uncurated protein sequences were annotated. The data obtained in the present study will aid physicians and researchers alike in curbing this bacterial infection.N/
Evaluation of Sofosbuvir (β-D-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine) as an inhibitor of Dengue virus replication
Get PDF© The Author(s) 2017. The version of record of this article, first published in [Scientific Reports], is available online at Publisher’s website: http://dx.doi.org/10.1038/s41598-017-06612-2We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine-5′-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.Government of Canada |CIHR| Institute of Infection and Immunit
Does antiretroviral treatment change HIV-1 codon usage patterns in its genes: a preliminary bioinformatics study
Get PDF© The Author(s) 2017. The version of record of this article, first published in [AIDS Research and Therapy], is available online at Publisher’s website: http://dx.doi.org/10.1186/s12981-016-0130-yBackground: Codon usage bias has been described for various organisms and is thought to contribute to the regulation of numerous biological processes including viral infections. HIV-1 codon usage has been previously shown to be different from that of other viruses and man. It is evident that the antiretroviral drugs used to restrict HIV-1 replication also select for resistance variants. We wanted to test whether codon frequencies in HIV-1 sequences from treatment-experienced patients differ from those of treatment-naive individuals due to drug pressure affecting codon usage bias. Results: We developed a JavaScript to determine the codon frequencies of aligned nucleotide sequences. Irrespective of subtypes, using HIV-1 pol sequences from 532 treatment-naive and 52 treatment-experienced individuals, we found that pol sequences from treatment-experienced patients had significantly increased AGA (arginine; p = 0.0002***) and GGU (glycine; p = 0.0001***), and decreased AGG (arginine; p = 0.0001***) codon frequencies. The same pattern was not observed when subtypes B and C sequences were analyzed separately. Additionally, irrespective of subtypes, using HIV-1 gag sequences from 524 treatment-naive and 54 treatment-experienced individuals, gag sequences from treatment-experienced patients had significantly increased CUA (leucine; p < 0.0001***), CAG (glutamine; p = 0.0006***), AUC (isoleucine; p < 0.0001***) and UCU (serine; p = 0.0005***), and decreased AUA (isoleucine; p = 0.0003***) and CAA (glutamine; p = 0.0006***) codon frequencies. Conclusion: Using pol and gag genes derived from the same HIV-1 genome, we show that antiretroviral therapy changed certain HIV-1 codon frequencies in a subtype specific way.Canadian Institutes for Health Research (CIHR
Why ignore expiry dates on cosmetics? A qualitative study of perceived risk and its implications for cosmetics producers and regulators
No full textThis is the peer reviewed version of the following article: [Wang, Y., Davies, G., Derbyshire, J., & Ullah, F. (2025). Why ignore expiry dates on cosmetics? A qualitative study of perceived risk and its implications for cosmetics producers and regulators, Risk Analysis, vol(issue), pages], which has been published in final form at [https://doi.org/10.1111/risa.70040]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.Consumers often use cosmetics long after their expiry date, despite the health risk. This paper aims to understand why and to suggest policy changes that can promote safer practices in cosmetics use. This is the first study to investigate risk perception in relation to expired cosmetics. Thirty-three semi-structured interviews with both cosmetics users and employees of cosmetics companies were conducted in the UK and China. Perceived risk theory was found to be a useful analytical lens. Eight risk factors emerged from the data, including two not previously identified. Combinations of risk were also found to be valuable in explaining consumer attitudes to cosmetic expiry dates, which suggests that perceived risk factors interact with each other to create an emergent perception of risk, requiring an integrated understanding. While physical, performance and self-brand connection risk can promote adherence to an expiry date, other risk factors such as financial and social risk can override such concerns, leading to the expiry date being ignored. Implications for suppliers’ and regulators’ policies and risk-communication strategies are identified that may help reduce the risks being taken by cosmetics users.Unfunde
Mean stability and between-session reliability of cycling biomechanics variables in elite pursuit cyclists
No full textThis is an Accepted Manuscript of an article published by Taylor & Francis in Sports Biomechanics on 03/03/2025, available online: https://doi.org/10.1080/14763141.2025.2471805The purpose of this study was to determine the number of crank revolutions required to obtain stable mean values of sagittal plane biomechanics variables, and the between-session reliability of these variables, whilst cyclists used an aerodynamic position. Eighteen elite cyclists completed a 3-min maximal bout on a cycling ergometer. Lower-limb kinematic and kinetic data were captured using 2D motion capture and force pedals. Raw data were filtered using a 4th order Butterworth low-pass filter (6 hz) and interpolated to 100 points per revolution. The middle 60 revolutions of each trial were extracted and 37 discrete and 15 time-series variables were calculated. Mean stability was assessed in all participants, and between-session reliability was analysed in a subset of 11 participants. Sequential averaging indicated more revolutions to stability than iterative intra-class correlation coefficients. Crank kinetics were more stable than joint kinematics and kinetics. For stable discrete and time-series variables, 30 and 38 revolutions are recommended, respectively. Between-day reliability for all variables was moderate to excellent, and good to excellent for crank kinetics and joint kinematics variables. Hip flexion-extension and ankle dorsiflexion kinetics were least reliable. Researchers and applied practitioners should consider these findings when planning, and interpreting results from, cycling biomechanics interventions.This study is part of a PhD programme of research jointly funded by British Cycling and Sheffield Hallam University
Defence Logistics: Enabling and Sustaining Successful Military Operations
No full textThe principal purpose of this chapter is to examine aspects of defence logistics, principally operational logistics, in order to shed light on some of the realities that give it its particular character, or at least some of its character. One possible approach is to analyse the defence supply chain and support chain using an analytical framework built around established through life support and supply chain paradigms, concepts, and ideas, drawn from the commercial sector. Another approach is to examine some specific characteristics of defence supply chains and support chains, some of their attributes, and some of their practical realities, and whilst doing so refer to the established paradigms, concepts, and ideas for comparison, but only where it seems appropriate to do so, in the hope that they might to some extent act as reference points by which defence logistics can be better understood by somebody not directly involved in its planning and delivery. One aim of this chapter is to examine the freedoms which defence departments and defence logisticians have in shaping their business, and the factors, or ‘realities’, which constrain them.Unfunde
Early years practitioners' and public health consultants' perspectives on the use of interactive electronic devices in young children: A qualitative study
Get PDF© 2025 The Author(s). Child: Care, Health and Development published by John Wiley & Sons Ltd.Background: Interactive electronic devices (IEDs) are ubiquitous in young children's lives. However, research on their impact on learning and development is still limited. The aim of this study was to understand the perspectives of early years practitioners (EYPs) and public health consultants (PHCs) on the use of IEDs in children aged 3–5. Methods: Using purposive sampling techniques, we recruited four EYPs and two PHCs from children's nurseries and a government organisation in the northwest of England. Semi‐structured interviews were used to collect data, which were audio‐recorded, transcribed verbatim and anonymised. Data were analysed using reflective thematic analysis. Results: EYPs and PHCs noted that although IEDs could negatively impact child development and behaviour, they could also aid in learning. EYPs expressed concerns about the impact of parents' own IED habits on children's communication and social skills. On the other hand, PHCs stressed that substituting outdoor play with the use of these devices could affect children's social and physical skills and reduce physical activity levels, which are crucial for development. Finally, both EYPs and PHCs agreed that there was a need to improve parents' and EYP's knowledge and to develop interactive interventions to promote an understanding of how IEDs should be used with young children. Conclusion: EYPs and PHCs acknowledge the potential advantages of using IEDs as a teaching tool for children. However, they have concerns about the long‐term effects on communication, social and physical skills and how children are impacted by their parents' use of these devices. To support policy statements, future research should offer further evidence of the benefits and harms of IED use.Unfunde
An assessment of burden associated with problem joints in children and adults with moderate or severe haemophilia A: analysis of the CHESS-Paediatrics and CHESS II cross-sectional studies
Get PDF© The Author(s) 2025.BACKGROUND: Clinical research has offered many definitions and fragmented perspectives of joint morbidity in haemophilia. As joint damage, pain and mobility impairment can be present without clinical record of persistent bleeding, a person-centric joint morbidity characterisation remained a priority for the haemophilia community, giving rise to the ‘problem joint’ concept. As diagnosing and managing joint morbidity is critical, the aim of this study was to analyse the holistic burden of problem joints in people with moderate or severe haemophilia A (HA). Data from the ‘Cost of Haemophilia in Europe: a Socioeconomic Survey’ (CHESS) cross-sectional studies were used. CHESS-Paediatrics included male paediatric patients (≤ 17 years) with congenital moderate or severe haemophilia, while CHESS II included adult males (≥ 18 years) of any severity. Both studies sought to collect detailed information on the clinical, economic and humanistic burden of haemophilia. Demographics, clinical outcomes, treatment regimen, adherence, physical activity, healthcare resource use and number of problem joints were evaluated and described by HA severity and number of problem joints (none, 1, ≥ 2).
RESULTS: In total, 1171 people with non-inhibitor moderate or severe HA from CHESS-Paediatrics (n = 703) and CHESS II (n = 468) were included in this analysis. Presence of problem joints was more prevalent among CHESS II participants (44%) than in CHESS-Paediatrics (14%). Around two-thirds (67%) of CHESS-Paediatrics and 39% of CHESS II participants received prophylactic factor VIII replacement therapy. The presence of chronic pain was greater in severe HA with ‘ ≥ 2’ problem joints in both cohorts. Clinical symptoms and bleed-related hospitalizations were more prevalent in the presence of problem joints regardless of HA severity in both cohorts.
CONCLUSIONS: This analysis of the CHESS population studies has expanded on previous work by examining the relevance of the problem joint measure of haemophilic morbidity and its associated burden. Adverse clinical symptoms and increased bleed-related hospitalizations were observed in the presence of problem joints in both children/adolescents and adults across HA severities. Use of person-centric characterizations of joint morbidity may improve analysis of long-term outcomes and lead to improvements in future haemophilia care.This work was sponsored by Roche. FN, MK and AN, employees of Roche, participated in the study design, interpretation of findings, and development of the manuscript. The wider CHESS II study was supported by unrestricted research grants from Sanofi, BioMarin and Takeda. The CHESS Paediatrics’ study was supported by research funding from Bayer, Roche, Swedish Orphan Biovitrum AB (Sobi), Novo Nordisk and Shire. Time reimbursement was provided to participating haemophilia healthcare practitioners according to relevant local fair market value rates. No specific benefits were provided to patients for the participation in the study
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