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Abstract Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense.

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease. Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition. Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3 + cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis. Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease., Competing Interests: OAC reports grants or contracts from Amplyx, Basilea, BMBF, Cidara, DZIF, EU-DG RTD (101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, and Scynexis; Consulting fees from Amplyx, Biocon, Biosys, Cidara, Da Volterra, Gilead, Matinas, MedPace, Menarini, Molecular Partners, MSG-ERC, Noxxon, Octapharma, PSI, Scynexis, Seres; Honoraria for lectures from Abbott, Al-Jazeera Pharmaceuticals, Astellas, Grupo Biotoscana/United Medical/Knight, Hikma, MedScape, MedUpdate, Merck/MSD, Mylan, Pfizer; Payment for expert testimony from Cidara; Participation on a Data Safety Monitoring Board or Advisory Board from Actelion, Allecra, Cidara, Entasis, IQVIA, Jannsen, MedPace, Paratek, PSI, Shionogi; A patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7); Other interests from DGHO, DGI, ECMM, ISHAM, MSG-ERC, and Wiley. PK reports grants or contracts from German Federal Ministry of Research and Education (BMBF) B-FAST (Bundesweites Forschungsnetz Angewandte Surveillance und Testung) and NAPKON (Nationales Pandemie Kohorten Netz, German National Pandemic Cohort Network) of the Network University Medicine (NUM) and the State of North Rhine-Westphalia; Consulting fees Ambu GmbH, Gilead Sciences, Mundipharma Resarch Limited, Noxxon N.V. and Pfizer Pharma; Honoraria for lectures from Akademie für Infektionsmedizin e.V., Ambu GmbH, Astellas Pharma, BioRad Laboratories Inc., European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, medupdate GmbH, MedMedia, MSD Sharp & Dohme GmbH, Pfizer Pharma GmbH, Scilink Comunicación Científica SC and University Hospital and LMU Munich; Participation on an Advisory Board from Ambu GmbH, Gilead Sciences, Mundipharma Resarch Limited and Pfizer Pharma; A pending patent currently reviewed at the German Patent and Trade Mark Office; Other non-financial interests from Elsevier, Wiley and Taylor & Francis online outside the submitted work. Authors HB, CC, OB, and MS are employed by the company Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany. Author DS is employed by the company Miltenyi Biomedicine GmbH, Bergisch Gladbach, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bonifacius, Tischer-Zimmermann, Santamorena, Mausberg, Schenk, Koch, Barnstorf-Brandes, Gödecke, Martens, Goudeva, Verboom, Wittig, Maecker-Kolhoff, Baurmann, Clark, Brauns, Simon, Lang, Cornely, Hallek, Blasczyk, Seiferling, Köhler and Eiz-Vesper.)

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans Far less is known about mammalian UPR(mt) signaling, although similar roles were assumed for central players, including CLPP To better understand the mammalian UPR(mt) signaling, we deleted CLPP in hearts of DARS2-deficient animals that show robust induction of UPR(mt) due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR(mt) in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR(mt) signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases., (© 2016 The Authors.)

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells by factor-driven induction., (Copyright © 2012 Elsevier Inc. All rights reserved.)