Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.

Following prevalence of myopia in a large Swiss military cohort over the last decade: where is the European "myopia boom"?

PURPOSE: Myopia prevalence is increasing globally, with the highest rates found in Asia. Data from European countries is scarce. We aimed to investigate whether the prevalence of myopia is rising in our meridians. METHODS: Data from male military conscripts for the recruitment period of 2008-2017 were retrospectively analyzed. Year of recruitment, conscripts' birth year, visual acuity, refractive status (spherical equivalent), and spectacle wear (yes/no) were available. RESULTS: The dataset contained data of a total of 355,657 male conscripts, who had been recruited in the years 2008 to 2017. The mean number of conscripts per year was 35,566 (MD = 35,440, SD = 1249), reaching a minimum number of 33,998 conscripts in 2017 and a maximum of 37,594 in 2011. Mean age at recruitment was 19.7 years (MD = 19.0 years, SD = 1.1 years). Overall, the number of conscripts wearing spectacles remained stable over the observation time; on average 29.6% (n = 10,540; MD = 10,472; SD = 492) of conscripts wore glasses at recruitment. Of 21.8% (n = 77,698) of conscripts, data on the refractive status was available: The mean spherical equivalent for both right and left eyes was -2.3D (MD = -2 D, SD = 2.4 D). No decrease in mean spherical equivalent per recruitment year was noted over the observation period. Estimated myopia prevalence reached an average of 27.5% (SD = 0.8%) and did not increase during the observation period. CONCLUSION: In summary, no change in spherical equivalent refractive errors of male Swiss army conscripts was found for the years 2008-2017. Equally, the percentage of spectacle wearers (MN = 29.6%) and estimated myopia prevalence (MN = 27.5%) did not significantly increase during the observation time. TRIAL REGISTRATION:  BASEC 2019-00060 (18/01/2019).

Proceedings of the 14th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals.

Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.

"Molecular Biology"-Pleonasm or Denotation for a Discipline of Its Own? Reflections on the Origins of Molecular Biology and Its Situation Today.

The disciplinary identity of molecular biology has frequently been called into question. Although the debates might sometimes have been more about creating or debunking myths, defending intellectual territory and the distribution of resources, there are interesting underlying questions about this area of biology and how it is conceptually organized. By looking at the history of molecular biology, its origins and development, I examine the possible criteria for its status as a scientific discipline. Doing so allows us to answer the title question in such a way that offers a reasonable middle ground, where molecular biology can be properly viewed as a viable interdisciplinary program that can very well be called a discipline in its own right, even if no strict boundaries can be established. In addition to this historical analysis, a couple of systematic issues from a philosophy of science perspective allow for some assessment of the current situation and the future of molecular biology.

Understanding immunity: an alternative framework beyond defense and strength.

In this paper we address the issue of how to think about immunity. Many immunological writings suggest a straightforward option: the view that the immune system is primarily a system of defense, which naturally invites the talk of strong immunity and strong immune response. Despite their undisputable positive role in immunology, such metaphors can also pose a risk of establishing a narrow perspective, omitting from consideration phenomena that do not neatly fit those powerful metaphors. Building on this analysis, we argue two things. First, we argue that the immune system is involved not only in defense. Second, by disentangling various possible meanings of 'strength' and 'weakness' in immunology, we also argue that such a construal of immunity generally contributes to the distortion of the overall picture of what the immune system is, what it does, and why it sometimes fails. Instead, we propose to understand the nature of the immune system in terms of contextuality, regulation, and trade-offs. We suggest that our approach provides lessons for a general understanding of the organizing principles of the immune system in health and disease. For all this to work, we discuss a wide range of immunological phenomena.

Minding the gut: extending embodied cognition and perception to the gut complex.

Scientific and philosophical accounts of cognition and perception have traditionally focused on the brain and external sense organs. The extended view of embodied cognition suggests including other parts of the body in these processes. However, one organ has often been overlooked: the gut. Frequently conceptualized as merely a tube for digesting food, there is much more to the gut than meets the eye. Having its own enteric nervous system, sometimes referred to as the "second brain," the gut is also an immune organ and has a large surface area interacting with gut microbiota. The gut has been shown to play an important role in many physiological processes, and may arguably do so as well in perception and cognition. We argue that proposals of embodied perception and cognition should take into account the role of the "gut complex," which considers the enteric nervous, endocrine, immune, and microbiota systems as well as gut tissue and mucosal structures. The gut complex is an interface between bodily tissues and the "internalized external environment" of the gut lumen, involved in many aspects of organismic activity beyond food intake. We thus extend current embodiment theories and suggest a more inclusive account of how to "mind the gut" in studying cognitive processes.

Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice.

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.

Emergency medicine in Switzerland: an analysis of physician workforce, gender equality and academics.

BACKGROUND: Globally, emergency medicine is continuously evolving and in numerous countries, societies and colleges help develop the specialty on a professional and academic level. However, there are countries, including Switzerland, where emergency medicine is not a fully recognised specialty and there is a historical gender gap. AIMS OF THE STUDY: It was the aim of this study to investigate the trends and developments in Swiss emergency medicine in terms of physician workforce, gender equality and academic posts over time. METHODS: In this observational longitudinal analysis, the number and gender distribution of Swiss Society of Emergency and Rescue Medicine (SSERM) members as well as SSERM-certified physicians were analysed in 2011, 2016 and 2021. Additionally, head and leading physicians of SSERM-certified emergency departments of category 1 and 2 were analysed in 2021 with special regard to gender distribution. Finally, an analysis of Swiss academic emergency medicine including Swiss academic tracks, professors in emergency medicine as well as committees, chairs and speakers of the annual SSERM conference was performed. RESULTS: From 2011 to 2021, there was an increase in SSERM members of 52% and a growing proportion of women from 26% to 35%. Similarly, there was a rise of 66% in physicians certified in in-hospital and 79% certified in prehospital emergency medicine. The proportion of women increased by 153% and 131%, respectively. In the analysed emergency departments, 69% of all head physicians were men whereas 50% of senior consultants and consultants with extended responsibility were women in 2021. Concerning academics, emergency medicine was a mandatory subject at all Swiss universities offering a master's degree in medical studies in 2021. However, 11 Swiss universities reported only six full professors, of whom only one was a woman, and three associate professors in emergency medicine in 2021. The analysis of the annual SSERM conferences from 2016 to 2019 revealed that men outnumbered women at every conference in terms of committees, chairs and speakers. CONCLUSIONS: The number of SSERM members and board-certified emergency physicians, women in particular, remarkably increased in 10 years. Equality appears to be within reach for clinical emergency physicians, but women continue to be underrepresented in academic positions, at scientific conferences and among professors. In Switzerland, academic emergency medicine appears to be lagging behind in view of the growing emergency physician and women workforce, which might complicate further progress in and development of Swiss emergency medicine on a scientific and professional level..

Unique acyl chains mark phosphoinositides for recycling.

Two recent complementary studies show that, after phospholipase C cleavage, the characteristic acyl chain composition of phosphoinositide-derived diacylglycerol funnels them back into the PI cycle.

Metabolic Labeling-Based Chemoproteomics Establishes Choline Metabolites as Protein Function Modulators.

Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions of choline and its metabolites, independent of their roles as choline lipid metabolism intermediates, remains limited. In addition to fundamental cellular physiology, this knowledge has implications for cancer biology because elevated choline metabolite levels are a hallmark of cancer. Here, we establish a mammalian choline metabolite-interacting proteome by utilizing a photocrosslinkable choline probe. To design this probe, we performed metabolic labeling experiments with structurally diverse choline analogues that resulted in the serendipitous discovery of a choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation steps. We demonstrate that phosphocholine inhibits the binding of one of the proteins identified, the attractive anticancer target p32, to its endogenous ligands and to the promising p32-targeting anticancer agent, Lyp-1. Our results reveal that choline metabolites play vital roles in cellular physiology by serving as modulators of protein function.

The microbial carbonate factory of Hamelin Pool, Shark Bay, Western Australia.

Microbialites and peloids are commonly associated throughout the geologic record. Proterozoic carbonate megafacies are composed predominantly of micritic and peloidal limestones often interbedded with stromatolitic textures. The association is also common throughout carbonate ramps and platforms during the Phanerozoic. Recent investigations reveal that Hamelin Pool, located in Shark Bay, Western Australia, is a microbial carbonate factory that provides a modern analog for the microbialite-micritic sediment facies associations that are so prevalent in the geologic record. Hamelin Pool contains the largest known living marine stromatolite system in the world. Although best known for the constructive microbial processes that lead to formation of these stromatolites, our comprehensive mapping has revealed that erosion and degradation of weakly lithified microbial mats in Hamelin Pool leads to the extensive production and accumulation of sand-sized micritic grains. Over 40 km2 of upper intertidal shoreline in the pool contain unlithified to weakly lithified microbial pustular sheet mats, which erode to release irregular peloidal grains. In addition, over 20 km2 of gelatinous microbial mats, with thin brittle layers of micrite, colonize subtidal pavements. When these gelatinous mats erode, the micritic layers break down to form platey, micritic intraclasts with irregular boundaries. Together, the irregular micritic grains from pustular sheet mats and gelatinous pavement mats make up nearly 26% of the total sediment in the pool, plausibly producing ~ 24,000 metric tons of microbial sediment per year. As such, Hamelin Pool can be seen as a microbial carbonate factory, with construction by lithifying microbial mats forming microbialites, and erosion and degradation of weakly lithified microbial mats resulting in extensive production of sand-sized micritic sediments. Insight from these modern examples may have direct applicability for recognition of sedimentary deposits of microbial origin in the geologic record.

When to Extend Monitoring of Anti-drug Antibodies for High-risk Biotherapeutics in Clinical Trials: an Opinion from the European Immunogenicity Platform.

The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Cyclotorsion Measurement on Scanning Laser Ophthalmoscopy Imaging Compared With Fundus Photography in Patients With Fourth Nerve Palsy and Healthy Controls: CySLO-IV Study.

BACKGROUND: The purpose of the study is to evaluate the agreement of the foveopapillary angle (FPA) on conventional fundus photography (c-FPA) with the FPA on scanning laser ophthalmoscopy (SLO) imaging (SLO-FPA) in patients with fourth nerve palsy and healthy controls (HCs). METHODS: The FPA was measured in both eyes of 25 patients and 25 HCs in synedra View (c-FPA) and with the integrated algorithm of the Heidelberg Spectralis OCT (SLO-FPA). The primary endpoint was the agreement of both measurements. Furthermore, we evaluated the influence of the eye tracker, the influence of fixation on objective torsion, and the FPA cutoff between patients and HCs. RESULTS: The mean SLO-FPA in patients (6/25 acquired palsies) was 11.3 ± 3.6° and 6.4 ± 2.1° in HCs. The mean c-FPA was 11.4 ± 4.0° and 5.8 ± 2.2°, respectively. The Bland-Altman plot of c-FPA vs SLO-FPA in patients and HCs shows no systematic bias (mean of -0.28°). Limits of agreement were -6.58 and 6.02°. Using the eye tracker had no systematic effect. There was no evidence for an immediate shift of torsion with change of fixation (24/25 patients and 23/25 HCs). Discrimination between patients and HCs by the SLO-FPA is very good with an area under the curve = 0.92 (95% confidence interval: 0.84-0.99). CONCLUSIONS: SLO-FPA measurement allows convenient and consistent assessment of objective cyclotorsion. There was no systematic bias in the difference between SLO-FPA and traditional c-FPA; thus, SLO-FPA is a valuable alternative to the commonly used c-FPA. Using the eye tracker is recommended for proper centering of the ring scan.

When metal-organic framework mediated smart drug delivery meets gastrointestinal cancers.

Cancers of the gastrointestinal tract constitute one of the most common cancer types worldwide and a ∼58% increase in the global number of cases has been estimated by IARC for the next twenty years. Recent advances in drug delivery technologies have attracted scientific interest for developing and utilizing efficient therapeutic systems. The present review focuses on the use of nanoscale MOFs (Nano-MOFs) as carriers for drug delivery and imaging purposes. In pursuit of significant improvements to current gastrointestinal cancer chemotherapy regimens, systems that allow multiple concomitant therapeutic options (polytherapy) and controlled release are highly desirable. In this sense, MOF-based nanotherapeutics represent a significant step towards achieving this goal. Here, the current state-of-the-art of interdisciplinary research and novel developments into MOF-based gastrointestinal cancer therapy are highlighted and reviewed.

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways.

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

The vaccinologist's "dirty little secret": a better understanding of structure-function relationships of viral immunogens might advance rational HIV vaccine design.

I will offer a conceptual analysis of different notions of structure and function of viral immunogens and of different structure-function relationships. My focus will then be on the mechanisms by which the desired immune response is induced and why strategies based on three-dimensional molecular antigen structures and their rational design are limited in their ability to induce the desired immunogenicity. I will look at the mechanisms of action of adjuvants (thus the wordplay with Janeway's "immunologist's dirty little secret"). Strategies involving adjuvants and other (more successful) vaccination strategies rely on taking into account activities and functions ("what is going on"), and not just the structures involved ("who is there"), in binding in a "lock and key" fashion. Functional patterns as well as other organizational and temporal patterns, I will argue, are crucial for inducing the desired immune response and immunogenicity. The 3D structural approach by itself has its benefits - and its limits, which I want to highlight by this philosophical analysis, pointing out the importance of structure-function relationships. Different functional aspects such as antigenicity, immunogenicity, and immunity need to be kept separate and cannot be reduced to three-dimensional structures of vaccines. Taking into account different notions of structure and function and their relationships might thus advance our understanding of the immune system and rational HIV vaccine design, to which end philosophy can provide useful tools.

All in One: Stimuli-Responsive, Efficient Mitotracking, and Single Source White Light Emission.

"All in one" type luminogens, possessing combined properties related to optical, materials, and biological implications, are of urgent demand today, mainly because of the combined application potential of such probes. To the best of our knowledge, until now, an "all in one" type white light emitter together with stimuli-responsive behavior and highly efficient mitochondrial-tracking ability has not been reported yet. In this contribution, for the first time, we have investigated a pair of luminogens exhibiting white light emission (CIE coordinates: 0.35, 0.35 (DPAEOA) and 0.29, 0.33 (DPAPMI)) with temperature-induced mechanochromic features of a centrosymmetrically packed probe (space group P-1). Most importantly, despite being neutral, our designed probe DPAEOA can specifically illuminate mitochondria with the highest Pearson coefficient value (0.93), which is rare, as almost all the commercially developed mitotrackers are cationic fluorophores. Thus, this study will pave a new avenue for the design of next generation "all in one" type organic luminogens exhibiting potential applications in notable optical, materials, and biological fields.

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3.

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.