Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration.

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene's numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

Molecular phenotyping uncovers differences in basic housekeeping functions among closely related species of hares (Lepus spp., Lagomorpha: Leporidae).

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

Manipulating mtDNA in vivo reprograms metabolism via novel response mechanisms.

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decrease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS.

The proto-oncometabolite fumarate binds glutathione to amplify ROS-dependent signaling.

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA.

Mitochondrial DNA (mtDNA) replication uses a simple core machinery similar to those of bacterial viruses and plasmids, but its components are challenging to unravel. Here, we found that, as in mammals, the single Drosophila gene for RNase H1 (rnh1) has alternative translational start sites, resulting in two polypeptides, targeted to either mitochondria or the nucleus. RNAi-mediated rnh1 knockdown did not influence growth or viability of S2 cells, but compromised mtDNA integrity and copy number. rnh1 knockdown in intact flies also produced a phenotype of impaired mitochondrial function, characterized by respiratory chain deficiency, locomotor dysfunction, and decreased lifespan. Its overexpression in S2 cells resulted in cell lethality after 5-9 days, attributable to the nuclearly localized isoform. rnh1 knockdown and overexpression produced opposite effects on mtDNA replication intermediates. The most pronounced effects were seen in genome regions beyond the major replication pauses where the replication fork needs to progress through a gene cluster that is transcribed in the opposite direction. RNase H1 deficiency led to an accumulation of replication intermediates in these zones, abundant mtDNA molecules joined by four-way junctions, and species consistent with fork regression from the origin. These findings indicate replication stalling due to the presence of unprocessed RNA/DNA heteroduplexes, potentially leading to the degradation of collapsed forks or to replication restart by a mechanism involving strand invasion. Both mitochondrial RNA and DNA syntheses were affected by rnh1 knockdown, suggesting that RNase H1 also plays a role in integrating or coregulating these processes in Drosophila mitochondria.

Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells.

RNA 3' polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNALeu(UUR) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.

Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits mitochondrial ROS overproduction.

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase.

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number.

Species-specific variation in mitochondrial genome tandem repeat polymorphisms in hares (Lepus spp., Lagomorpha, Leporidae) provides insight into their evolution.

The non-coding regions of the mitochondrial DNAs (mtDNAs) of hares, rabbits, and pikas (Lagomorpha) contain short (∼20 bp) and long (130-160 bp) tandem repeats, absent in related mammalian orders. In the presented study, we provide in-depth analysis for mountain hare (Lepus timidus) and brown hare (L. europaeus) mtDNA non-coding regions, together with a species- and population-level analysis of tandem repeat variation. Mountain hare short tandem repeats (SRs) as well as other analyzed hare species consist of two conserved 10 bp motifs, with only brown hares exhibiting a single, more variable motif. Long tandem repeats (LRs) also differ in sequence and copy number between species. Mountain hares have four to seven LRs, median value five, while brown hares exhibit five to nine LRs, median value six. Interestingly, introgressed mountain hare mtDNA in brown hares obtained an intermediate LR length distribution, with median copy number being the same as with conspecific brown hare mtDNA. In contrast, transfer of brown hare mtDNA into cultured mtDNA-less mountain hare cells maintained the original LR number, whereas the reciprocal transfer caused copy number instability, suggesting that cellular environment rather than the nuclear genomic background plays a role in the LR maintenance. Due to their dynamic nature and separation from other known conserved sequence elements on the non-coding region of hare mitochondrial genomes, the tandem repeat elements likely to represent signatures of ancient genetic rearrangements. clarifying the nature and dynamics of these rearrangements may shed light on the possible role of NCR repeated elements in mitochondria and in species evolution.

Glucose modulates respiratory complex I activity in response to acute mitochondrial dysfunction.

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an "emergency shutdown" system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction.

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model.

Foam cells are a pathological feature present at all stages of atherosclerosis. Foam cells develop from monocytes that enter the nascent atheroma and subsequently ingest modified low density lipoproteins (LDL). The regulation of this process has previously been studied in vitro using cultured macrophage fed modified LDL. We used our existing in vitro model of transendothelial migration (TEM) to study this process in a more physiologically relevant setting. In our model, monocytes undergo TEM across a primary endothelial monolayer into an underlying three-dimensional collagen matrix in the presence of 20% human serum. Foam cells were detected by Oil Red O staining for intracellular lipid droplets. We demonstrate that sub-endothelial monocytes can develop into foam cells within 48 h of TEM across TNF-α activated endothelium, in the absence of additional lipids. Our data indicate a role for both monocyte-endothelial interactions and soluble factors in the regulation of foam cell development, including oxidation of LDL in situ from lipid present in culture medium following TNF-α stimulation of the endothelial cells. Our study provides a simple model for investigating foam cell development in vitro that mimics cell migration in vivo, and demonstrates the critical role of inflammation in regulating early atherogenic events.

Ultrasound-guided perineural circumferential median nerve block with and without prior dextrose 5% hydrodissection: a prospective randomized double-blinded noninferiority trial.

BACKGROUND: Ultrasound-guided perineural peripheral nerve block using a hydrodissection technique may reduce the risk of accidental intravascular local anesthetic (LA) injection. In this prospective randomized double-blind study, we tested the hypothesis that median nerve block effectiveness is not reduced when circumferential perineural hydrodissection with dextrose 5% in water (D5W) precedes LA injection. METHODS: Patients scheduled for hand surgery were randomized to receive an ultrasound-guided median nerve block at the elbow to achieve circumferential perineural spread with either 6 mL of D5W followed by 6 mL of LA (lidocaine 1.5% with epinephrine 1:200,000) (D5W-LA group) or with 6 mL of LA alone (LA group). The primary outcome was onset time of successful anesthesia defined by a complete abolition of light touch sensation for the index finger. RESULTS: Data from 95 patients were analyzed: 43 in the D5W-LA group and 52 in the LA group. Noninferiority tests were significant (all P < 0.05) for a critical limit of 7 minutes between D5W-LA and LA groups for onset time of the primary criterion, light touch block at index finger (mean ± SD, respectively: 23.9 ± 7.4 and 22.0 ± 7.9 minutes; 95% confidence interval [CI], -5.9 to 2.1 minutes), and for cold block at index fingertip, sensory blocks at thenar eminence, and motor block. Success rate at 30 minutes (defined as complete abolition for cold and light touch at index finger) was noted in 100% and 98.1% (95% CI, -6% to 10%) and 95.2% and 96.2% (95% CI, -13% to 9%) of patients for the D5W-LA and the LA groups. CONCLUSION: Performing an ultrasound-guided perineural circumferential hydrodissection with D5W into which LA is injected leaves nerve block outcome unchanged. The assumption that this procedure may reduce the risk of intravascular injection and systemic toxicity remains to be demonstrated.

Nano-sized cosmetic formulations or solid nanoparticles in sunscreens: a risk to human health?

Personal care products (PCP) often contain micron- or nano-sized formulation components, such as nanoemulsions or microscopic vesicles. A large number of studies suggest that such vesicles do not penetrate human skin beyond the superficial layers of the stratum corneum. Nano-sized PCP formulations may enhance or reduce skin absorption of ingredients, albeit at a limited scale. Modern sunscreens contain insoluble titanium dioxide (TiO2) or zinc oxide (ZnO) nanoparticles (NP), which are efficient filters of UV light. A large number of studies suggest that insoluble NP do not penetrate into or through human skin. A number of in vivo toxicity tests, including in vivo intravenous studies, showed that TiO2 and ZnO NP are non-toxic and have an excellent skin tolerance. Cytotoxicity, genotoxicity, photo-genotoxicity, general toxicity and carcinogenicity studies on TiO2 and ZnO NP found no difference in the safety profile of micro- or nano-sized materials, all of which were found to be non-toxic. Although some published in vitro studies on insoluble nano- or micron-sized particles suggested cell uptake, oxidative cell damage or genotoxicity, these data are consistent with those from micron-sized particles and should be interpreted with caution. Data on insoluble NP, such as surgical implant-derived wear debris particles or intravenously administered magnetic resonance contrast agents suggest that toxicity of small particles is generally related to their chemistry rather than their particle size. Overall, the weight of scientific evidence suggests that insoluble NP used in sunscreens pose no or negligible risk to human health, but offer large health benefits, such as the protection of human skin against UV-induced skin ageing and cancer.

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: skin sensitisation.

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the 'gold standard' test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.

A randomized trial to determine the minimum effective lidocaine volume for median nerve block using hydrodissection.

Ultrasound-guided hydrodissection with 5% dextrose in water (DW5) creates a peri-nervous compartment, separating the nerve from the neighboring anatomical structures. The aim of this randomized study was to determine the minimum volume of lidocaine 2% with epinephrine 1:200,000 required when using this technique to achieve an effective median nerve block at the elbow in 95% of patients (MEAV95). Fifty-two patients scheduled for elective hand surgery received an ultrasound-guided circumferential perineural injection of 4 ml DW5 and an injection of local anesthetic (LA) following a biased coin up-and-down sequential allocation method. A successful block was defined as a light touch completely suppressed on the two distal phalanges of the index finger within a 30-min evaluation period. The MEAV95 of lidocaine 2% with epinephrine was 4 ml [IQR 3.5-4.0]. Successful median nerve block was obtained in 38 cases (82.6%) with median onset time of 20.0 [10.0-21.2] minutes (95% CI 15-20). The analgesia duration was 248 [208-286] minutes (95% CI 222-276). Using an ultrasound-guided hydrodissection technique with DW5, the MEAV95 to block the median nerve at the elbow with 2% lidocaine with epinephrine was 4 ml [IQR 3.5-4.0]. This volume is close to that usually recommended in clinical practice.Trial registration clinicaltrials.gov. NCT02438657, Date of registration: May 8, 2015.

Prevention of SARS-CoV-2 transmission during a large, live, indoor gathering (SPRING): a non-inferiority, randomised, controlled trial.

BACKGROUND: Mass indoor gatherings were banned in early 2020 to prevent the spread of SARS-CoV-2. We aimed to assess, under controlled conditions, whether infection rates among attendees at a large, indoor gathering event would be similar to those in non-attendees, given implementation of a comprehensive prevention strategy including antigen-screening within 3 days, medical mask wearing, and optimised ventilation. METHODS: The non-inferiority, prospective, open-label, randomised, controlled SPRING trial was done on attendees at a live indoor concert held in the Accor Arena on May 29, 2021 in Paris, France. Participants, aged 18-45 years, recruited via a dedicated website, had no comorbidities, COVID-19 symptoms, or recent case contact, and had had a negative rapid antigen diagnostic test within 3 days before the concert. Participants were randomly allocated in a 2:1 ratio to the experimental group (attendees) or to the control group (non-attendees). The allocation sequence was computer-generated by means of permuted blocks of sizes three, six, or nine, with no stratification. The primary outcome measure was the number of patients who were SARS-CoV-2-positive by RT-PCR test on self-collected saliva 7 days post-gathering in the per-protocol population (non-inferiority margin <0·35%). This trial is registered with ClinicalTrials.gov, NCT04872075. FINDINGS: Between May 11 and 25, 2021, 18 845 individuals registered on the dedicated website, and 10 953 were randomly selected for a pre-enrolment on-site visit. Among 6968 who kept the appointment and were screened, 6678 participants were randomly assigned (4451 were assigned to be attendees and 2227 to be non-attendees; median age 28 years; 59% women); 88% (3917) of attendees and 87% (1947) of non-attendees complied with follow-up requirements. The day 7 RT-PCR was positive for eight of the 3917 attendees (observed incidence, 0·20%; 95% CI 0·09-0·40) and three of the 1947 non-attendees (0·15%; 0·03-0·45; absolute difference, 95% CI -0·26% to 0·28%), findings that met the non-inferiority criterion for the primary endpoint. INTERPRETATION: Participation in a large, indoor, live gathering without physical distancing was not associated with increased SARS-CoV-2-transmission risk, provided a comprehensive preventive intervention was implemented. FUNDING: French Ministry of Health. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.