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  • Mouth rinsing and ingesting unpleasant salty or bitter solutions after heavy-intensity cycling does not influence sprint performance or knee-extensor force in trained cyclists

    Gray, Edward A.; Cavaleri, Rocco; Siegler, Jason C.; University of Chester; Western Sydney University; Arizona State University (Human Kinetics, 2024-12-28)
    PURPOSE: The present study investigated the effect of unpleasant salty or bitter tastes on cycling sprint performance and knee-extensor force characteristics in different fatigue states. METHODS: Following a familiarization session, 11 trained male cyclists completed 3 experimental trials (salty, bitter, and water) in a randomized crossover order. In each trial, participants cycled at 85% of the respiratory compensation point for 45 minutes and then, after a 5-minute rest, completed a 1-minute sprint. Muscle-force characteristics were assessed using 2 knee-extensor maximal voluntary contractions immediately before, between, and after the cycling efforts. Participants mouth-rinsed and ingested 25 mL of test solution (salty, bitter, and water) immediately before each maximal voluntary contractions and the 1-minute sprint. RESULTS: There were no significant differences in mean and peak power output during the 1-minute sprint between conditions (mean power: 528 [71] W, 524 [70] W, and 521 [80] W in the water, salt, and bitter conditions, respectively). Muscle-force production was impaired in all conditions after the heavy-intensity cycling, evidenced by a decline in maximum force production (P = .01, effect size = 0.32) and 100- to 200-millisecond impulse (P = .04, effect size = 0.27). However, there were no significant differences between conditions in maximal force or impulse measures at rest or after exercise. CONCLUSION: These data question whether unpleasant tastes can influence muscle-force production and do not support that they may be used as an ergogenic aid for a cycling sprint performed under fatigued conditions.
  • Evidence-based nursing in action: A focused ethnographic case study of knowledge use in acute care

    Ominyi, Jude; Eze, Ukpai; Alabi, Adewale; Nwedu, Aaron; University of Suffolk; University of Chester; Coventry University; David Umahi Federal University of Health Sciences, Nigeria (Elsevier, 2025-07-05)
    Introduction: Evidence-based practice (EBP) plays a crucial role in improving care outcomes in critical care settings. However, its integration into nursing practice remains challenging due to organisational hierarchies, workload pressures, and uneven access to formal knowledge. This study explores how critical care nurses access, use, and integrate knowledge, with a focus on how organisational culture, leadership, and team dynamics influence EBP implementation across two acute care sites in England. Methods: The study adopted a focused ethnographic design, guided by Spradley’s Developmental Research Sequence. Data were collected over eight months through 210 hours of non-participant observation, 36 semi-structured interviews, and document analysis. Analysis was supported using NVivo 12, applying iterative thematic coding. Reflexivity and member checking were used to ensure analytical rigour and trustworthiness. Results: Five central themes were identified: access to formal guidelines, the role of peer learning, organisational culture and hierarchy, the value of experiential knowledge, and barriers to EBP. While both sites demonstrated reliance on blended sources of knowledge, they differed in how organisational factors shaped access and engagement. Site A showed stronger support through mentorship, simulation-based learning, and active leadership. In contrast, Site B was marked by rigid structures and informal, inconsistent communication of evidence. Conclusion: Critical care nurses draw on both formal evidence and informal knowledge, but the conditions of their work environment influence how and when this knowledge is used. Investment in leadership, protected learning time, and inclusive communication practices is essential to support sustainable engagement with EBP in high-pressure settings.
  • Implementing Evidence‐Based Practice in Critical Care Nursing: An Ethnographic Case Study of Knowledge Use

    Ominyi, Jude; Eze, Ukpai; Agom, David; Alabi, Adewale; Nwedu, Aaron; University of Suffolk; University of Chester; University of Bedfordshire; Coventry University; David Umahi Federal University of Health Sciences (2025-07-04)
    Aim: To explore how critical care nurses access, negotiate and apply knowledge in high‐pressure clinical environments, focusing on organisational, cultural and leadership factors influencing evidence‐based practice implementation in acute hospital settings. Design: A focused ethnographic collective case study was conducted across two contrasting critical care units in England. Methods: Methods included non‐participant observation (56 sessions), semi‐structured interviews (36 participants) and document review. Spradley's Developmental Research Sequence guided data generation and analysis. Data were collected over an eight‐month period (February to September 2022). Findings: Five major themes were identified: sources of knowledge and acquisition strategies; institutional and hierarchical influences on knowledge use; role of experiential knowledge and clinical intuition; challenges to evidence‐based practice implementation; and strategies for integrating knowledge into practice. Organisational structures, leadership engagement, mentorship and access to updated digital resources were key enablers of evidence‐based practice. Barriers included workload pressures, inconsistent guideline dissemination and hierarchical cultures. Adaptive blending of formal evidence, clinical experience and intuition characterised effective knowledge negotiation at the bedside. Conclusion: Knowledge use in critical care nursing is a dynamic, relational process shaped by leadership, organisational culture and systemic pressures. The availability of evidence alone is insufficient; visible leadership, peer learning, protected educational time and valuing of experiential knowledge are critical to embedding evidence‐based practice into routine practice. Implications for Patient Care: Strengthening organisational systems, investing in nurse manager development, expanding simulation‐based learning and legitimising experiential knowledge are vital strategies to enhance evidence‐based critical care. Impact: This study provides actionable insights for healthcare leaders, educators and policymakers seeking to optimise evidence‐based practice adoption in high‐acuity clinical environments and improve patient outcomes. Reporting Method: The Consolidated Criteria for Reporting Qualitative Research checklist guided reporting. No Patient or Public Involvement: Patients and the public were not involved in the design, conduct, reporting or dissemination of this research.
  • SCCS Opinion on Biphenyl-2-ol and Sodium 2-biphenylolate used in cosmetic products (CAS/EC No. 90-43-7/201-993-5 and 132-27-4/205-055-6)– SCCS/1669/24

    Bernauer (rapporteur), Ulrike; Bodin, Laurent; Chaudhry, Qasim; Coenraads, Pieter Jan; Ezendam, Janine; Gaffet, Eric; Galli, Corrado L.; Panteri, Eirini; Rogiers, Vera; Rousselle, Christophe; et al. (Elsevier, 2025-06-04)
    ‘Biphenyl-2-ol’ and ‘Sodium 2-biphenylolate’ (CAS/EC No. 90-43-7/201-993-5 and 132-27-4/205-055-6) are known with the INCI names ‘o-Phenylphenol’ and Sodium ‘o-Phenylphenate’, respectively, as cosmetic ingredients. The use of o-Phenylphenol as a preservative is currently authorised in entry 7 of Annex V to the Cosmetics Regulation (EC) No.1223/2009, with a maximum concentration of 0.15 % (as phenol) in leave-on and 0.2 % (as phenol) in rinse-off cosmetic products. The Scientific Committee on Consumer Safety (SCCS) adopted an opinion on o-Phenylphenol (OPP), Sodium o-Phenylphenate (SOPP), and Potassium o-Phenylphenate (POPP) (SCCS/1555/15) in June 2015, later revised on 15 December 2015, with the following conclusion: ‘Based on the information provided, no conclusions of safe use can be drawn for Sodium o-Phenylphenate and Potassium o-Phenylphenate’. In 2018, the SCCS released an addendum to the above scientific opinion, specifically addressing Sodium o-Phenylphenate, Potassium o-Phenylphenate, and MEA o-Phenylphenate. The SCCS concluded that ‘Due to the lack of relevant information, the SCCS is unable to answer the question regarding the safe use level of sodium-OPP, potassium-OPP, and MEA-OPP. In the SCCS's view, a direct comparison between the safety of o-Phenylphenate (OPP) and its three compounds cannot be made’. The conclusions of this opinion resulted in amending entry 7 of Annex V to the Cosmetics Regulation, removing from the list the previously authorised OPP salts. It should be noted that o-Phenylphenol, Sodium and Potassium o-Phenylphenate are active ingredients in broad-spectrum fungicides surface biocides. Under EU biocidal Regulation (EU) 528/2012, o-Phenylphenol has been evaluated for the different product types (PTs) such as PT 1, PT 2, PT4, PT 6 as a preservative ranging from 0.1 to 0.5 % w/w. The European Risk Assessment Committee (RAC) of ECHA issued in December 2022 an opinion recommending among others a classification for o-Phenylphenol as ‘Carcinogen of Category 2’. Following the RAC opinion, the European Commission may propose a classification for o-Phenylphenol as a ‘Carc.2’ (CLP Regulation Annex VI entry). According to Article 15(1) of the Cosmetics Regulation ‘the use in cosmetic products of substances classified as CMR substances, of category 2, under Part 3 of Annex VI to Regulation (EC) No 1272/2008 shall be prohibited. However, a substance classified in category 2 may be used in cosmetic products where the substance has been evaluated by the SCCS and found safe for use in cosmetic products’. In view of these provisions, regulatory measures must be adopted by the Commission services within 15 months of the classification as CMR 1A or 1B of the substance(s) concerned in Part 3 of Annex VI to Regulation (EC) No 1272/2008. In December 2023, the Commission services received a dossier to defend the safe use of o-Phenylphenol, as well as its sodium salt (CAS/EC No. 90-43-7/201-993-5 and 132-27-4/205-055-6) as preservatives in cosmetic products according to Article 15(1) of the Cosmetics Regulation 1223/2009. The Commission, therefore, requests the SCCS to carry out a safety assessment on these ingredients in view of the information provided.
  • Association between 2D landing biomechanics, isokinetic muscle strength and asymmetry in females using novel, task specific metrics based on ACL injury mechanisms

    Oxendale, Chelsea; Smith, Grace; Liverpool John Moores University; University of Chester (Public Library of Science, 2025-07-01)
    This study investigated the relationship between isokinetic muscle strength metrics, landing biomechanics, and their asymmetries, in females. Twenty-three female team sport athletes completed unilateral forward drop landings, and isokinetic muscle strength assessment of the knee extensors and flexors, on both limbs. Discrete two-dimensional kinematics of the trunk, hip, knee, and ankle in the sagittal and frontal plane and peak GRF were recorded during the drop landings. Novel, task-specific isokinetic strength metrics related to the landing task, such as peak concentric and eccentric torque, angle specific torque (AST), functional range and traditional/functional ratios were quantified. Asymmetry for kinematic and muscle strength data were quantified based on the individual variability of the task and the population mean and smallest worthwhile change. Functional concentric flexor range explained 15–18% of the variance in peak frontal trunk (P = 0.003) and hip motion (P = 0.007) and 22% in peak frontal knee motion (P = 0.005), when combined with the functional flexion ratio. Peak eccentric extensor torque explained 13–14% of the variance in peak sagittal hip (P = 0.014) and knee (P = 0.009) motion. Asymmetry in concentric extensor AST explained 28% of the variance in peak knee frontal plane asymmetry (P = 0.010), however the direction of asymmetry was rarely present on the same side for kinematic and strength variables. Novel and task specific isokinetic strength metrics explained small but significant variances in sagittal and frontal plane landing kinematics and asymmetry, which have previously been related to ACL injury risk.
  • Harried and stressful or hurried and rewarding? A study of young Norwegians’ active leisure lives

    Johansen, Patrick Foss; Green, Ken; Thurston, Miranda; Universitetet i Innlandet (University of Inland Norway); University of Chester (Taylor & Francis, 2025-06-17)
    Amidst the backdrop of a ‘speed-up society’, this study examines how youngsters navigate their discretionary time while juggling educational demands, social networks, family obligations, and leisure activities. Through semi-structured interviews with 41 sports-active Norwegian youngsters, the study analysed their leisure activities and experiences of time pressure. The findings revealed that, contrary to perceptions of a harried existence, young Norwegians generally perceived their busy leisure lives to be rewarding rather than stressful. Their engagement in active leisure, especially sports, was seen as a counterbalance to the stresses of educational demands. Leisure, it seems, acted as an enclave for mental and physical recreation, aiding in managing school-related stress. Their ongoing involvement in sport underscore the importance of early life experiences in shaping youths’ leisure choices. It also serves to remind us of the potential for busy leisure lives to be experienced as hurried and rewarding rather than straightforwardly harried and stress-inducing.
  • Achieving COVID-19 herd immunity in Bangladesh

    Mary, Rabeya Mashferat; Alam, Falguni; Miranda, Adriana Viola; Lucero-Prisno, Don Eliseo; Bulbul, Md Mofijul Islam; University of Chester; North South University, Dhaka; Global Health Focus Asia, Bandung; London School of Hygiene and Tropical Medicine; Ministry of Health and Family Welfare, Bangladesh (Wiley, 2023-06-06)
    Achieving herd immunity against COVID-19 through vaccination is a global target, including in lower resource settings. Despite the challenges and limitations, Bangladesh has achieved its target of vaccinating 70% of its population, with good vaccine coverage among refugees, remote population, and women. This can be attributed to the evidence-informed adaptability and collaborative approaches of the program. Yet some challenges remain, including dependence on donors and the need to ramp up vaccination among the underserved communities. This article discusses the factors contributing to the achievements of the COVID-19 vaccination program of Bangladesh and the remaining challenges that should be addressed by the government to ensure the sustainability of the program and inform future vaccination efforts.
  • The Impact of Sleep Deprivation on Brain Fog, Cognitive Decline, and Cardiovascular Risk in Young Adults

    Younas, Aimen; Vayolipoyil, Sarath; Raghib, Shaheera; Bano, Sher; Wandala, Abali; Khan, Azmir Ali; Amin, Areej; Asim Khan, Aima; Muhammad Ali, Syed; Iqbal, Javed; et al. (Springer Nature, 2025-05-08)
    Introduction Sleep deprivation is an escalating public health concern among young adults, as it impairs cognitive function and increases the risk of cardiovascular disease (CVD). Existing studies have linked chronic sleep deficiencies to mental confusion, reduced cognitive performance, and early signs of cognitive decline. Research also indicates that inadequate sleep contributes to metabolic dysregulation and autonomic system instability, both of which elevate cardiovascular risk. However, the overall effects of sleep deprivation on cognitive function and cardiovascular markers in young adults require further exploration. This study aims to examine the relationship between sleep deprivation, brain fog, early cognitive decline, and cardiovascular risk factors in this population. Methods This cross-sectional study involved 300 participants aged 18-30 from Pakistan and various other countries. Participants were selected using non-probability purposive sampling. Data were collected using four validated instruments: the Pittsburgh Sleep Quality Index, the Cognitive Failures Questionnaire, the Mini-Mental State Examination, and the Perceived Stress Scale. Data analysis was conducted using IBM SPSS Statistics for Windows, Version 26.0 (Released 2019; IBM Corp., Armonk, NY, USA), applying chi-square tests, independent sample t-tests, ANOVA, Pearson correlation, and logistic regression to assess associations between sleep deprivation, cognitive performance, and cardiovascular outcomes. Results Participants with shorter sleep durations had significantly higher scores in cognitive failures (p < 0.01) and perceived stress (p < 0.01). Poor sleep quality was associated with reduced cognitive performance (r = -0.114, p < 0.05), and it also increased the likelihood of developing cardiovascular risk factors. Participants with a family history of CVD exhibited significantly higher cognitive failure scores (t = 5.540, p < 0.001). Furthermore, a decline in sleep quality was associated with increased cardiovascular risk (B = 0.035, p = 0.019), although sleep disorders were not significantly influenced by sleep quality deterioration (B = 0.012, p = 0.400). Employment status and smoking habits were also found to impact both sleep quality and cognitive function (p < 0.01). Conclusions This study highlights the adverse impact of insufficient sleep on cognitive function and cardiovascular health in young adults aged 18-30. Poor sleep quality is associated with increased cognitive errors, heightened stress levels, and a greater risk of cardiovascular issues. These findings emphasize the importance of targeted health interventions aimed at improving sleep hygiene and lifestyle behaviors to reduce the risk of early cognitive decline and cardiovascular conditions. Future research should employ longitudinal designs and objective sleep tracking to strengthen causal inferences. [Abstract copyright: Copyright © 2025, Younas et al.]
  • (Un)sustainability of the elite parasport development system in a Least Developed Country: an investigation of Cambodian para-Athletic career pathways

    Nagata, Shinichi; Bloyce, Daniel; Sato, Takahiro; Yoneyama, Haruka; Nishiyama, Naoki; Boomgaard, Pepijn; Okade, Yoshinori (Taylor and Francis, 2025-06-09)
    Purpose: Establishing athletic career pathways is important for the sustainable development of elite parasport, but there is a paucity of literature on parasport athletic career pathways in economically disadvantaged, Least Developed Countries. The current study investigated how para-athletes, coaches, and administrators in Cambodia experience their athletic careers. Methodology: Individual, semi-structured interviews were conducted with four para-athletics coaches, six para-athletics athletes, and two parasport administrators in Cambodia. The transcripts were analyzed with thematic analysis. Findings and practical implications: Key themes indicated that the lack of parasport equipment limits new athlete recruitment, the lack of a positive career outlook after athletic retirement is a cause of distress among athletes, and specialized parasport coaches are lacking. These problems suggest alack of sustainability. The Cambodian government could invest in a clearer retirement preparation system for para-athletes. In this respect, retired para-athletes could continue contributing to parasport development in the country as elite and grassroots-level coaches. Research contribution: This is the first study examining the experience of para-athletes, coaches, and administrators in a Least Developed Country. Our findings identified recruitment, retirement preparation, and coach training as major areas for development. Value: The findings would assist evidence-based policy making for more sustainable development of elite parasport.
  • An Investigation of Hydrocolloids Film Forming Ability on a Vertically Rotating Disc

    Miah, Salim; McMillan, Alison; Al-Assaf, Saphwan; University of Hertfordshire; Wrexham University; University of Chester (Bangladesh Academy of Sciences, 2023-12-30)
    Hydrocolloids are widely used in the food industry to perform variety of functions such as coatings, thickening, emulsifying, stabilizing and edible films. Their functionality for a given application are underpinned by the molecular weight, shape, and conformation in aqueous solution. The film forming ability of selected hydrocolloids, different in shape (rod, random coil and spherical) and/or conformation in aqueous solution were investigated experimentally and numerically on a vertically rotating disc. These include: xanthan, pectin, carboxymethyl cellulose (CMC) and gum arabic. The Laser scan method was used for the measurement of film thickness of the respective the hydrocolloids. The Volume of Fluid (VOF) Computational Fluid Dynamics (CFD) modelling approach was used in the numerical model. The variation in film formation at different concentrations has been observed to ascertain a trend. Both the experimental and simulation results revealed that the film formation depends on the molecular structure of the hydrocolloid while viscosity and rotating speed significantly influenced the film thickness. Xanthan showed higher film formation ability compared to the other hydrocolloids due to its higher viscosity. It was interesting to note that the film formation ability by CMC was significantly higher than pectin though pectin was five times more viscous than CMC. Gum arabic exhibited the lowest viscosity but formed almost the same film thickness on the disc as pectin despite being twenty times less viscous. Increasing CMC concentration from 0.5% to 1% resulted in increasing its viscosity and the film thickness. The film thickness increased at the disc rotating speed of 6 rpm as compared to 3 rpm. The simulation results were in good agreement with the experimental data.
  • Author Correction: Split intein-mediated selection of cells containing two plasmids using a single antibiotic

    Palanisamy, Navaneethan; Degen, Anna; Morath, Anna; Ballestin, Jara Ballestin; Juraske, Claudia; Öztürk, Mehmet Ali; Sprenger, Georg A; Youn, Jung-Won; Schamel, Wolfgang W; Di Ventura, Barbara; et al. (Nature Research, 2020-01-09)
    Correction to: Nature Communications https://doi.org/10.1038/s41467-019-12911-1, published online 31 October 2019.
  • C-terminal eYFP fusion impairs Escherichia coli MinE function

    Palanisamy, Navaneethan; Öztürk, Mehmet Ali; Di Ventura, Barbara; University of Freiburg; University of Heidelberg (OpenRxiv (bioRxiv), 2020-01-10)
    The Escherichia coli Min system plays an important role in the proper placement of the septum ring (Z-ring) at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator together with the membrane-bound ATPase MinD, which results in MinD having a concentration gradient with maxima at the poles and minimum at mid-cell. MinC, the direct inhibitor of the Z-ring initiator protein FtsZ, forms a complex with MinD at the membrane, thus mirroring MinD polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. The existence of the oscillations was revealed by performing time-lapse microscopy with fluorescently-labeled Min proteins. These fusion proteins have been since then widely used to study properties of the Min system. Here we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that the eYFP compromises MinE ability to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. Taken together, our results indicate that this fusion is functionally impaired and should be used with caution in cell biological studies.
  • Influenza A virus encoding secreted Gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins

    Eckert, Nadine; Wrensch, Florian; Gärtner, Sabine; Palanisamy, Navaneethan; Goedecke, Ulrike; Jäger, Nils; Pöhlmann, Stefan; Winkler, Michael; German Primate Center, Göttingen (Public Library of Science, 2014-05-19)
    Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1-3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.
  • SARS-CoV-2 Omicron variants and their susceptibility towards Monoclonal Antibodies in a Swedish cohort during 2022-23, studied by next-generation sequencing

    Haars, Jonathan; Palanisamy, Navaneethan; Wallin, Frans; Mölling, Paula; Lindh, Johan; Sundqvist, Martin; Ellström, Patrik; Kaden, René; Lennerstrand, Johan; Uppsala University; University of Chester; Örebro University Hospital; Örebro University; SciLifeLab, Clinical Genomics Uppsala (Prepints.org, 2023-09-12)
    Monoclonal antibodies (mAbs) are an important treatment option for COVID-19 caused by SARS-CoV-2, especially in immunosuppressed patients. However, this treatment option can become ineffective due to mutations in the SARS-CoV-2 genome, mainly in the receptor binding domain (RBD) of the spike (S) protein. In the present study 7950 SARS-CoV-2 positive samples from the Uppsala and Örebro regions of central Sweden collected between March 2022 and May 2023 were whole-genome sequenced using next-generation sequencing, mainly with the Nanopore sequencing method. Pango lineages were determined and all single nucleotide polymorphism (SNP) mutations that occurred in these samples were identified. The dominant sublineages changed over time and mutations conferring resistance to currently available mAbs became common. Notable ones are R346T and K444T mutations in the RBD that confer significant resistance against tixagevimab and cilgavimab mAbs. Further, mutations conferring a high-fold resistance to bebtelovimab, such as the K444T and V445P mutations, were also observed in the samples. This study highlights that resistance mutations have over time rendered currently available mAbs ineffective against SARS-CoV-2 in most patients. Therefore, there is a need for continued surveillance of resistance mutations and the development of new mAbs that target more conserved regions of the RBD.
  • Identification of functional Npu DnaE and gp41-1 inteins split in three fragments

    Weis, Daniel; Palanisamy, Navaneethan; Ballestin, Jara Ballestin; Öztürk, Mehmet Ali; Di Ventura, Barbara; University of Freiburg (OpenRxiv (bioRxiv), 2023-03-06)
    Inteins are special proteins that auto-catalytically carry out a protein splicing reaction. Due to their ability to post-translationally modify target proteins in vitro and in vivo, they are used in different applications, ranging from protein purification to the construction of Boolean logic gates. So far inteins have been found to be either encoded by a single gene (contiguous inteins) or by two separate ones (split inteins). Previously, it has been shown that the contiguous Ssp and Rma DnaB inteins and the split Npu DnaE intein could be artificially split in three fragments and retain functionality. Here we report the identification of novel split sites within the N-terminal fragments of the Npu DnaE and gp41-1 split inteins that lead to synthetic functional three-piece versions of these inteins. These variants contribute to the toolkit of three-piece inteins that could be used in biotechnological applications based on highly-fragmented inteins.
  • Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin

    Palanisamy, Navaneethan; Ballestin, Jara Ballestin; Di Ventura, Barbara; ViraTherapeutics GmbH (OpenRxiv (bioRxiv), 2021-02-04)
    We 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.
  • An inducible AraC that responds to blue light instead of arabinose

    Romano, Edoardo; Baumschlager, Armin; Akmeriç, Emir Bora; Palanisamy, Navaneethan; Houmani, Moustafa; Schmidt, Gregor; Öztürk, Mehmet Ali; Ernst, Leonard; Khammash, Mustafa; Di Ventura, Barbara; et al. (OpenRxiv (bioRxiv), 2020-07-14)
    In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.
  • SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

    Kokkula, Chakradhar; Palanisamy, Navaneethan; Ericstam, Malin; Lennerstrand, Johan; Uppsala University; Genome Valley Shameerpet, Hyderabad; University of Heidelberg (Springer Nature, 2016-07-04)
    There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.
  • Biophysical Studies on HCV 1a NS3/4A Protease and Its Catalytic Triad in Wild Type and Mutants by the In Silico Approach

    Palanisamy, Navaneethan; Lennerstrand, Johan; University of Heidelberg; Uppsala University (Springer Nature, 2016-06-16)
    The hepatitis C virus (HCV), of the family flaviviridae, is one of the major causes of chronic liver diseases. Until the year 2012, HCV infections were treated using PEG-interferon and ribavirin combinations, which have a low cure rate and severe side effects. Currently, many direct-acting antivirals (DAAs) are available, e.g. protease inhibitors, NS5A and polymerase inhibitors. These drugs have proven to be efficient in interferon-free treatment combinations and capable of enhancing the cure rate to above 90 %. Unlike PEG-interferon and ribavirin combinations, DAAs select for resistance in HCV. The R155K mutation in the HCV was found to resist all the currently available protease inhibitors. Here, we studied biophysical parameters like pocket (cavity) geometries and stabilizing residues of HCV 1a NS3/4A protease in wild type and mutants. We also studied HCV 1a NS3/4A protease’s catalytic residues: their accessibility, energy, flexibility and binding to Phase II oral protease inhibitor vedroprevir (GS-9451), and compared these parameters between wild type and mutant(s). All these studies were performed using various bioinformatics tools (e.g. Swiss-PdbViewer and Schrödinger’s Maestro) and web servers (e.g. DoGSiteScorer, SRide, ASA-View, WHAT IF, elNémo, CABS-flex, PatchDock and PLIP). From our study, we found that introduction of R155K, A156T or D168A mutation to wild-type NS3/4A protease increases the pocket’s volume, surface (in the R155K mutant, surface decreases), lipo surface and depth and decreases the number of stabilizing residues. Additionally, differences in catalytic residues’ solvent accessibility, energy, root-mean-square deviation (RMSD) and flexibility between wild type and mutants might explain changes in the protease activity and the resistance to protease inhibitors.
  • 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

    Akaberi, Dario; Bergfors, Assar; Kjellin, Midori; Kameli, Nader; Lidemalm, Louise; Kolli, Bhavya; Shafer, Robert W; Palanisamy, Navaneethan; Lennerstrand, Johan; Uppsala University; Maastricht University Medical Center; Stanford University; University of Heidelberg; University of Freiburg; et al. (Taylor & Francis, 2018-10-05)
    Background: 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.

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