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Energy-converting NADH: ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular energy metabolism. Bacterial complex I is generally composed of 14 different subunits, seven of which are membranous and the other seven are globular proteins. They are encoded by the nuo-operon, whose gene order is strictly conserved in bacteria. The operon starts with nuoA encoding a membranous subunit followed by genes encoding globular subunits. To test the idea that NuoA acts as a seed to initiate the assembly of the complex in the membrane, we generated mutants that either lacked nuoA or contain nuoA at a different position within the operon. To enable the detection of putative assembly intermediates, the globular subunit NuoF and the membranous subunit NuoM were individually decorated with the fluorescent protein mCherry. Deletion of nuoA led to the assembly of an inactive complex in the membrane containing NuoF and NuoM. Re-arrangement of nuoA within the nuo-operon led to a slightly diminished amount of complex I in the membrane that was fully active. Thus, nuoA but not its distinct position in the operon is required for the assembly of E. coli complex I. Furthermore, we detected a previously unknown assembly intermediate in the membrane containing NuoM that is present in greater amounts than complex I., (Copyright © 2022 Elsevier B.V. All rights reserved.)

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application's ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at https://itrex.kitz-heidelberg.de., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Energy converting NADH:ubiquinone oxidoreductase, complex I, is the first enzyme of respiratory chains in most eukaryotes and many bacteria. The complex comprises a peripheral arm catalyzing electron transfer and a membrane arm involved in proton-translocation. In Escherichia coli, the peripheral arm features a non-covalently bound flavin mononucleotide and nine iron-sulfur (Fe/S)-clusters. Very little is known about the incorporation of the Fe/S-clusters into the E. coli complex I. ErpA, an A-type carrier protein is discussed to act as a Fe/S-cluster carrier protein. To contribute to the understanding of ErpA for the assembly of E. coli complex I, we analyzed an erpA knock-out strain. Deletion of erpA decreased the complex I content in cytoplasmic membranes to approximately one third and the NADH oxidase activity to one fifth. EPR spectroscopy showed the presence of all Fe/S-clusters of the complex in the membrane but only in minor quantities. Sucrose gradient centrifugation and native PAGE revealed the presence of a marginal amount of a stable and fully assembled complex extractable from the membrane. Thus, ErpA is not essential for the assembly of complex I but its absence leads to a strong decrease of a functional complex in the cytoplasmic membrane due to a major lack of all EPR-detectable Fe/S-clusters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax., (© 2016 by The American Society of Hematology.)

Sirolimus (Rapammune, rapamycin, RAPA) is a potent immunosuppressive drug that reduces renal transplant rejection. Hyperlipidemia is a significant side effect of sirolimus treatment, and frequently leads to cardiovascular disease. This study was undertaken to determine the repeatability, reversibility, and dose dependence of the plasma lipid and apolipoprotein altering effects of sirolimus, and to elucidate the mechanism by which sirolimus induces hypertriglyceridemia in some renal transplant patients. Six patients with renal allografts maintained on cyclosporine A and prednisone were selected on the basis of their previous hyperlipidemic response to short term (14 days) sirolimus administration. For longer-term treatment, each patient was started on 10 mg/day sirolimus and continued as tolerated for 42 days to reinduce hyperlipidemia. Timed blood samples were analyzed for lipid, apolipoprotein, and sirolimus levels. During sirolimus administration, mean total plasma cholesterol increased from 214 mg/dl to 322 mg/dl (+50%; range 25-92%); LDL-cholesterol levels followed a similar pattern. Mean triglyceride level rose from 227 to 432 mg/dl (+95%; range 9-254%). ApoB-100 concentration rose from 124 to 160 mg/dl (+28%; P < 0.05). ApoC-III level increased from 28.9 to 55.5 mg/dl, +92%; (P < 0.013). These lipid and apolipoprotein changes were found to be repeatable, reversible, and dose dependent. [(13)C(4)]palmitate metabolic studies in four patients with hypertriglyceridemia indicated that the free fatty acid pool was expanded by sirolimus treatment (mean = 42.3%). Incorporation of [(13)C(4)]palmitate into triglycerides of VLDL, IDL, and LDL was decreased 38.3%, 42,1%, and 38.4%, respectively, by sirolimus treatment of these patients. These results suggest that sirolimus alters the insulin signaling pathway so as to increase adipose tissue lipase activity and/or decrease lipoprotein lipase activity, resulting in increased hepatic synthesis of triglyceride, increased secretion of VLDL, and increased hypertriglyceridemia.

Study Objectives: To study the in vitro effects of the serotonin2 (5-HT2) receptor agonist 1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in skeletal muscle specimens from malignant hyperthermia-susceptible (MHS) and normal (MHN) patients following pretreatment with the 5-HT2 receptor antagonist ritanserin., Design: Prospective study., Setting: Malignant hyperthermia (MH) laboratory at a university hospital., Patients: 41 patients undergoing in vitro contracture test for diagnosis of MH susceptibility., Interventions: Skeletal muscle biopsies in adult patients were performed with a 3-in-1 nerve block with 40 ml prilocaine 1%. In children, general anesthesia was induced with 50 micrograms/kg alfentanil intravenously (i.v.) and 2 to 2.5 micrograms/kg propofol i.v. and maintained with a continuous infusion of propofol (< or = 150 micrograms/kg/min) and nitrous oxide (66%) in oxygen., Measurements and Main Results: Patients were first classified as MHS or MHN by the in vitro contracture test according to the European MH protocol. Surplus muscle specimens of 21 MHS and 20 MHN patients were used in this study. At first, DOI was added to the organ bath at a concentration of 0.02 mM. In the second part of the study, muscle specimens were preincubated with ritanserin 0.01 mM for 10 minutes before DOI 0.02 mM was added to the bath. Muscle specimens from all patients developed contractures after administration of DOI. The onset of contractures was significantly faster in MHS muscles, and the magnitude of contracture was significantly greater than in MHN. The muscle twitch decreased significantly in both groups after DOI. After pretreatment with ritanserin, start of contracture was significantly delayed in MHS muscles. MHN muscles failed to develop contractures. The maximum level of contracture was significantly reduced in MHS. Muscle twitch decreased also in both MHS and MHN groups., Conclusions: The findings may indicate that stimulation of 5-HT2 receptors is involved in MH induction. Furthermore, 5-HT2 receptor antagonists could possibly be effective in preventing MH. Additional studies are required to determine if administration of 5-HT2 receptor antagonists could be of additional value in the treatment or prevention of anesthetic-induced MH.