No Fault Found: The Root Cause

No Trouble Found (NTF) has been discussed for several years [1]. An NTF occurs when a device fails at the board/system level and that failure cannot be confirm by the component supplier. There are several explanations for why NTFs occur, including: device complexity; inability to create system level hardware/software transactions which uncover hard to find defects; different environments during testing (power, thermal, noise). More recently a new concept, No Fault Found (NFF), has emerged. A NFF represents a defect which cannot be detected by any known means so far. The premise is that at some point the defect will be exposed - most likely at a customer site when the device is in a system. Given that we looking for a defect that we know nothing about and are theoretically undetectable it will be interesting to see what the panel has to say about the nature of these defects and how we intend to find them

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.Fil: Bedez, Claire. Université de Strasbourg; FranciaFil: Lotz, Christophe. Université de Strasbourg; FranciaFil: Batisse, Claire. Université de Strasbourg; FranciaFil: Broeck, Arnaud Vanden. Université de Strasbourg; FranciaFil: Stote, Roland H.. Université de Strasbourg; FranciaFil: Howard, Eduardo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Pradeau-Aubreton, Karine. Université de Strasbourg; FranciaFil: Ruff, Marc. Université de Strasbourg; FranciaFil: Lamour, Valérie. Université de Strasbourg; Franci

Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex

DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Identification of Mycobacteria in Routine Clinical Practice

Background: Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed. Methodology: We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10 5 colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25M. avium and 12 non-tuberculosis clinical isolates with identification scores $2 within 2.5 hours. Conclusions: Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heatinactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories i

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Functional and structural study of type II DNA topoisomerases and their post-translational modifications

Les ADN topoisomérases de type 2 (Top2s) régulent la topologie de l’ADN dans de nombreux processus cellulaires. Ce sont des cibles majeures dans le traitement face au cancer et a certains agents pathogènes. Cependant l’administration des molécules anti Top2s peut entrainer des effets secondaires. Des connaissances structurales supplémentaires sur les Top2s sont donc nécessaires afin de développer des molécules plus spécifiques. Nous avons dans un premier temps étudie la régulation de l’activité de la TopIIα par les modifications post-traductionnelles (PTMs). L’identification des PTMs a mis en avant une lysine dont la modification perturbe le couplage structure/fonction a été mise en évidence. La caractérisation de la TopIIα déphosphorylée a montré que ces modifications modulent son activité catalytique. Les PTMs affectent des positions clés, et ont un impact majeur sur la relation structure-fonction de TopIIα permettant de réguler l’activité lors du cycle catalytique. Une revue de la littérature a permis d’observer des différences dans les PTMs suivant les phases du cycle cellulaire. Enfin, nous avons obtenu les structures de la TopIIα humaine et la TopII procaryote, l’ADN Gyrase, montrant des différences au niveau des connexions entre les domaines. L’étude des activités a montré que ces connexions sont importantes pour la communication allostérique entre les domaines. L’ensemble de ces informations apportent des éléments sur la modulation de l’activité des TopIIs expliquée par leurs structures et les PTMs.Type 2 DNA topoisomerases (Top2s) regulate DNA topology in many cellular processes. They are major targets in the treatment of cancer and against certains pathogens. However, the administration of anti-Top2 molecules can cause side effects. Additional structural knowledge on TopIIs is therefore necessary in order to develop more specific molecules. We first studied the regulation of TopII by post-translational modifications (PTMs). The identification of PTMs highlighted a lysine whose modification disrupts the structure/function coupling. The characterization of dephosphorylated TopIIα showed that these modifications influence catalytic activities. PTMs affect key positions and have a major impact on the structure-function relationship of TopIIα allowing to regulate the activity during the catalytic cycle. A review of the literature showed differences in PTMs according to cell status. In a second project we obtained the structures of human TopIIα and prokaryotic TopII, DNA gyrase, showing differences in the connections between the domains. The study of the activities showed that these connections are important for the allosteric communication between the domains.All this information provides elements on the modulation of the activity of the TopIIs explained by their structure and PTMs

Etude fonctionnelle et structurale des ADN topoisomérases de type II et de leurs modifications post-traductionnelles

Type 2 DNA topoisomerases (Top2s) regulate DNA topology in many cellular processes. They are major targets in the treatment of cancer and against certains pathogens. However, the administration of anti-Top2 molecules can cause side effects. Additional structural knowledge on TopIIs is therefore necessary in order to develop more specific molecules. We first studied the regulation of TopII by post-translational modifications (PTMs). The identification of PTMs highlighted a lysine whose modification disrupts the structure/function coupling. The characterization of dephosphorylated TopIIα showed that these modifications influence catalytic activities. PTMs affect key positions and have a major impact on the structure-function relationship of TopIIα allowing to regulate the activity during the catalytic cycle. A review of the literature showed differences in PTMs according to cell status. In a second project we obtained the structures of human TopIIα and prokaryotic TopII, DNA gyrase, showing differences in the connections between the domains. The study of the activities showed that these connections are important for the allosteric communication between the domains.All this information provides elements on the modulation of the activity of the TopIIs explained by their structure and PTMs.Les ADN topoisomérases de type 2 (Top2s) régulent la topologie de l’ADN dans de nombreux processus cellulaires. Ce sont des cibles majeures dans le traitement face au cancer et a certains agents pathogènes. Cependant l’administration des molécules anti Top2s peut entrainer des effets secondaires. Des connaissances structurales supplémentaires sur les Top2s sont donc nécessaires afin de développer des molécules plus spécifiques. Nous avons dans un premier temps étudie la régulation de l’activité de la TopIIα par les modifications post-traductionnelles (PTMs). L’identification des PTMs a mis en avant une lysine dont la modification perturbe le couplage structure/fonction a été mise en évidence. La caractérisation de la TopIIα déphosphorylée a montré que ces modifications modulent son activité catalytique. Les PTMs affectent des positions clés, et ont un impact majeur sur la relation structure-fonction de TopIIα permettant de réguler l’activité lors du cycle catalytique. Une revue de la littérature a permis d’observer des différences dans les PTMs suivant les phases du cycle cellulaire. Enfin, nous avons obtenu les structures de la TopIIα humaine et la TopII procaryote, l’ADN Gyrase, montrant des différences au niveau des connexions entre les domaines. L’étude des activités a montré que ces connexions sont importantes pour la communication allostérique entre les domaines. L’ensemble de ces informations apportent des éléments sur la modulation de l’activité des TopIIs expliquée par leurs structures et les PTMs