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Simon K. Krebs Marlitt Stech Felix Jorde Nathanaël Rakotoarinoro Franziska Ramm Sophie Marinoff Sven Bahrke Antje Danielczyk Doreen A. Wüstenhagen Stefan Kubick 《International journal of molecular sciences》2022,23(22)
Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required. 相似文献
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Jessica L. Braun Mia S. Geromella Sophie I. Hamstra Holt N. Messner Val A. Fajardo 《International journal of molecular sciences》2021,22(21)
It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35–37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA. 相似文献
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John G. Fisher reja Benan Marija Kosec Sophie Vernay Daniel Rytz 《Journal of the American Ceramic Society》2008,91(5):1503-1507
(K0.5 Na0.5 )NbO3 (KNN) is a potential lead-free replacement for Pb(Zr, Ti)O3 (PZT) piezoceramics, but its piezoelectric properties are inferior to those of PZT. By growing single crystals of KNN, it may be possible to improve the piezoelectric properties. Recently, single crystals of KNN were grown by the solid-state crystal growth (SSCG) method, but the crystals were very porous. In this paper, a method of growing dense single crystals by SSCG in a hot press will be described. (110)-oriented single seed crystals of KTaO3 were buried in KNN powder, with 0.5 mol% of K4 CuNb8 O23 added as a sintering aid. After consolidation by uniaxial and isostatic pressing, the tablets were hot pressed in a two-stage treatment. During hot pressing, dense single crystals of KNN grew using the KTaO3 seed crystal as a template. The effect of hot pressing on single crystal growth will be discussed. 相似文献
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Sophie Couture Thomas G. Brown Jacques Tremblay N.M.K. Ng Ying Kin Marie Claude Ouimet Louise Nadeau 《Accident; analysis and prevention》2010,42(1):307-312
A first driving while impaired by alcohol (DWI) conviction is a key opportunity to identify offenders who are at high risk for recidivism. Detection of alcohol use disorder (AUD) is a major target of current DWI assessments. However, offenders frequently underreport their alcohol consumption, and use of biomarkers has been proposed as a more objective indicator. Among the best established are aspartate aminotranferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), mean corpuscular red blood cell volume (MCV), carbohydrate-deficient transferrin (CDT), and thiamine. To our knowledge, no research has directly verified whether AUD biomarkers predict DWI recidivism status. Using a cross-sectional design, this study tested three hypotheses related to the utility of biomarkers in DWI assessment.