| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Pineda-Pampliega, Javier, et al.
(författare)
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Influence of climatic conditions on the link between oxidative stress balance and landfill utilisation as a food resource by white storks
- 2023
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Ingår i: Science of the Total Environment. - 0048-9697. ; 903
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Tidskriftsartikel (refereegranskat)abstract
- Landfills have played a significant role in the recovery of white storks (Ciconia ciconia) populations across various European countries. While there is ample information about the populational-level effects, there is a lack of knowledge regarding the individual effects of using this food resource for feeding nestlings. This study aims to assess the nutritional status and oxidative stress balance of nestlings with varying degrees of exposure to landfill-provided food This study aims to assess the nutritional status and oxidative stress balance of nestlings with different use of landfill-provided food. Nestlings fed with food foraged by breeding pairs from landfills exhibited better nutritional status compared to individuals located farther from landfills. This can be attributed to a higher ingestion rate, resulting in increased plasmatic values of cholesterol, triglycerides, and HDL in plasma. However, the oxidative stress balance varied across different years, with individuals raised in 2014 showing higher values of Vitamin E and lower values of LDH compared to those raised in 2013. Furthermore, the impact of landfills on certain oxidative stress parameters also depended on the year of study. In 2013, the Total Antioxidant Capacity (TAC) of plasma showed a positive correlation with the distance to landfills, while the concentration of Malondialdehyde (MDA), an indicator of lipid peroxidation, exhibited a negative correlation. These findings suggest that the use of landfills as a food resource has a consistently positive effect on the nutritional status of white stork nestling. However, the relationship with oxidative stress is highly dependent on the climatic conditions of each year, emphasizing the importance of considering these factors when evaluating the use of landfills as a food resource.
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| 3. |
- Aguirre Rivera, Javier, 1989-, et al.
(författare)
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Real-time measurements of aminoglycoside effects on protein synthesis in live cells
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 118:9
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Tidskriftsartikel (refereegranskat)abstract
- The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.
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| 4. |
- Aguirre Rivera, Javier, 1989-
(författare)
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Tracking single molecules in uncharted territory : A single-molecule method to study kinetics in live bacteria
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.
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| 5. |
- Ferrando, Carlos, et al.
(författare)
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Individualised, perioperative open-lung ventilation strategy during one-lung ventilation (iPROVE-OLV) : a multicentre, randomised, controlled clinical trial
- 2024
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Ingår i: The Lancet Respiratory Medicine. - : Elsevier. - 2213-2600 .- 2213-2619. ; 12:3, s. 195-206
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Tidskriftsartikel (refereegranskat)abstract
- Background It is uncertain whether individualisation of the perioperative open-lung approach (OLA) to ventilation reduces postoperative pulmonary complications in patients undergoing lung resection. We compared a perioperative individualised OLA (iOLA) ventilation strategy with standard lung-protective ventilation in patients undergoing thoracic surgery with one-lung ventilation. Methods This multicentre, randomised controlled trial enrolled patients scheduled for open or video-assisted thoracic surgery using one-lung ventilation in 25 participating hospitals in Spain, Italy, Turkey, Egypt, and Ecuador. Eligible adult patients (age >= 18 years) were randomly assigned to receive iOLA or standard lung-protective ventilation. Eligible patients (stratified by centre) were randomly assigned online by local principal investigators, with an allocation ratio of 1:1. Treatment with iOLA included an alveolar recruitment manoeuvre to 40 cm H2O of end-inspiratory pressure followed by individualised positive end-expiratory pressure (PEEP) titrated to best respiratory system compliance, and individualised postoperative respiratory support with high-flow oxygen therapy. Participants allocated to standard lungprotective ventilation received combined intraoperative 4 cm H2O of PEEP and postoperative conventional oxygen therapy. The primary outcome was a composite of severe postoperative pulmonary complications within the first 7 postoperative days, including atelectasis requiring bronchoscopy, severe respiratory failure, contralateral pneumothorax, early extubation failure (rescue with continuous positive airway pressure, non-invasive ventilation, invasive mechanical ventilation, or reintubation), acute respiratory distress syndrome, pulmonary infection, bronchopleural fistula, and pleural empyema. Due to trial setting, data obtained in the operating and postoperative rooms for routine monitoring were not blinded. At 24 h, data were acquired by an investigator blinded to group allocation. All analyses were performed on an intention-to-treat basis. This trial is registered with ClinicalTrials.gov, NCT03182062, and is complete. Findings Between Sept 11, 2018, and June 14, 2022, we enrolled 1380 patients, of whom 1308 eligible patients (670 [434 male, 233 female, and three with missing data] assigned to iOLA and 638 [395 male, 237 female, and six with missing data] to standard lung-protective ventilation) were included in the final analysis. The proportion of patients with the composite outcome of severe postoperative pulmonary complications within the first 7 postoperative days was lower in the iOLA group compared with the standard lung-protective ventilation group (40 [6%] vs 97 [15%], relative risk 0 center dot 39 [95% CI 0 center dot 28 to 0 center dot 56]), with an absolute risk difference of -9 center dot 23 (95% CI -12 center dot 55 to -5 center dot 92). Recruitment manoeuvre-related adverse events were reported in five patients. Interpretation Among patients subjected to lung resection under one-lung ventilation, iOLA was associated with a reduced risk of severe postoperative pulmonary complications when compared with conventional lung-protective ventilation. Funding Instituto de Salud Carlos III and the European Regional Development Funds. Copyright (c) 2023 Elsevier Ltd. All rights reserved.
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| 6. |
- Sabantsev, Anton, et al.
(författare)
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Spatiotemporally controlled generation of NTPs for single-molecule studies
- 2022
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Ingår i: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469. ; 18:10, s. 1144-
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Tidskriftsartikel (refereegranskat)abstract
- Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.
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| 7. |
- Seefeldt, A. Carolin, et al.
(författare)
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Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
- 2021
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 433:10
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Tidskriftsartikel (refereegranskat)abstract
- Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA(Phe) on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA(fMet). By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA(Phe), underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.
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| 8. |
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The Seventeenth Data Release of the Sloan Digital Sky Surveys : Complete Release of MaNGA, MaStar, and APOGEE-2 Data
- 2022
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Ingår i: Astrophysical Journal Supplement Series. - : Institute of Physics (IOP). - 0067-0049 .- 1538-4365. ; 259:2
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Tidskriftsartikel (refereegranskat)abstract
- This paper documents the seventeenth data release (DR17) from the Sloan Digital Sky Surveys; the fifth and final release from the fourth phase (SDSS-IV). DR17 contains the complete release of the Mapping Nearby Galaxies at Apache Point Observatory (MaNGA) survey, which reached its goal of surveying over 10,000 nearby galaxies. The complete release of the MaNGA Stellar Library accompanies this data, providing observations of almost 30,000 stars through the MaNGA instrument during bright time. DR17 also contains the complete release of the Apache Point Observatory Galactic Evolution Experiment 2 survey that publicly releases infrared spectra of over 650,000 stars. The main sample from the Extended Baryon Oscillation Spectroscopic Survey (eBOSS), as well as the subsurvey Time Domain Spectroscopic Survey data were fully released in DR16. New single-fiber optical spectroscopy released in DR17 is from the SPectroscipic IDentification of ERosita Survey subsurvey and the eBOSS-RM program. Along with the primary data sets, DR17 includes 25 new or updated value-added catalogs. This paper concludes the release of SDSS-IV survey data. SDSS continues into its fifth phase with observations already underway for the Milky Way Mapper, Local Volume Mapper, and Black Hole Mapper surveys.
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