A Temperature-sensitive Mutant of Escherichia Coli Affected in the Alpha Subunit of RNA Polymerase

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44\sp\circC. The thermostability at 44\sp\circC of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about four fold at 37\sp\circC. The role of the alpha dimer in the structure and function of RNA polymerase is discussed. In addition during the course of this study a new procedure for the purification of E. coli RNA polymerase was developed. This method is rapid, convenient, and useful for the preparation of enzyme from 1-5 grams of cells in two days. The ease and speed of this method allowed the rapid characterization of the mutant enzyme. This system should also find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase

Nanoparticles for live cell microscopy: A surface-enhanced Raman scattering perspective.

Surface enhanced Raman scattering (SERS) nanoparticles are an attractive alternative to fluorescent probes for biological labeling because of their photostability and multiplexing capabilities. However, nanoparticle size, shape, and surface properties are known to affect nanoparticle-cell interactions. Other issues such as the formation of a protein corona and antibody multivalency interfere with the labeling properties of nanoparticle-antibody conjugates. Hence, it is important to consider these aspects in order to validate such conjugates for live cell imaging applications. Using SERS nanoparticles that target HER2 and CD44 in breast cancer cells, we demonstrate labeling of fixed cells with high specificity that correlates well with fluorescent labels. However, when labeling live cells to monitor surface biomarker expression and dynamics, the nanoparticles are rapidly uptaken by the cells and become compartmentalized into different cellular regions. This behavior is in stark contrast to that of fluorescent antibody conjugates. This study highlights the impact of nanoparticle internalization and trafficking on the ability to use SERS nanoparticle-antibody conjugates to monitor cell dynamics

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

A Temperature-Sensitive Mutant of Escherichia Coli Affected in the Alpha Subunit of RNA Polymerase

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min. on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44°C. The thermostability at 44°C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37°C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed

A Rapid Method for the Purification of RNA Polymerase Holoenzyme From Escherichia Coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gell filtration chromatography gave 95% pure holoenzyme. The enzyme kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures

Nanoparticles for live cell microscopy: A surface-enhanced Raman scattering perspective.

Surface enhanced Raman scattering (SERS) nanoparticles are an attractive alternative to fluorescent probes for biological labeling because of their photostability and multiplexing capabilities. However, nanoparticle size, shape, and surface properties are known to affect nanoparticle-cell interactions. Other issues such as the formation of a protein corona and antibody multivalency interfere with the labeling properties of nanoparticle-antibody conjugates. Hence, it is important to consider these aspects in order to validate such conjugates for live cell imaging applications. Using SERS nanoparticles that target HER2 and CD44 in breast cancer cells, we demonstrate labeling of fixed cells with high specificity that correlates well with fluorescent labels. However, when labeling live cells to monitor surface biomarker expression and dynamics, the nanoparticles are rapidly uptaken by the cells and become compartmentalized into different cellular regions. This behavior is in stark contrast to that of fluorescent antibody conjugates. This study highlights the impact of nanoparticle internalization and trafficking on the ability to use SERS nanoparticle-antibody conjugates to monitor cell dynamics

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges

Considerations for MESF-bead based assignment of absolute fluorescence values to nanoparticles and extracellular vesicles by flow cytometry

Flow cytometry is a promising technique to characterize nanoparticles (NPs) and extracellular vesicles (EVs). However, the majority of reported experiments, use arbitrary units to indicate fluorescence intensity. This hampers comparison of results from different laboratories and different platforms. We investigated the advised use of calibrated molecules of equivalent soluble fluorophores (MESF)-beads for assignment of absolute fluorescence to NPs and EVs. Firstly, we evaluated the use of two different FITC MESF bead sets as calibrators on three different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Secondly, NPs and biological 4T1 mammary carcinoma-EVs were analyzed using the BD Influx and their fluorescence signals calibrated by using different sets of FITC and PE MESF beads. Although fluorescence calibration, using bright calibrators designed for cellular flow cytometry, makes inter-platform comparison possible for fluorescently labeled cells and brightly labeled particles, but the uncertainty of the currently available calibrators, which are far out of the fluorescence range of the sub-micron particles, hampers a reliable assignment of absolute MESF numbers based on extrapolation into the dim fluorescence range. Our results illustrate the need for calibration materials specifically designed for NPs and EVs to enable a reliable assignment of absolute fluorescence values in the lower fluorescent ranges