Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The picornavirus, encephalomyocarditis virus, employs a type 2 IRES present in its 5’ untranslated region (5’UTR) and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of encephalomyocarditis virus (EMCV) IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contact initiator tRNA (tRNA<sub>i</sub>) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNA<sub>i</sub> is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins uS13 and uS19. The structural analysis accounts for numerous previously reported biochemical studies on type 2 IRES and shows how type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, including type 1 viral IRESs.
Science Journals
The outflow tract (OFT) of the heart carries blood away from the heart into the great arteries. During embryogenesis, the OFT divides to form the aorta and pulmonary trunk, creating the double circulation present in mammals. Defects in this area account for one-third of all congenital heart defect cases. Here, we present comprehensive transcriptomic data on the developing OFT at two distinct time points (embryonic and fetal) and its adult derivatives, the aortic valves, and use spatial transcriptomics to define the distribution of cell populations. We uncover that distinctive embryonic signatures persist in adult cells and can be used as labels to retrospectively attribute relationships between cells separated by a large timescale. Single-cell regulatory network inference identifies GATA6, a transcription factor linked to common arterial trunk and bicuspid aortic valve, as a key regulator of valve precursor cells. Its downstream network reveals candidate drivers of human cardiac defects and illuminates the molecular mechanisms of both normal and pathological valve development. Our findings define the cellular and molecular signatures of the human OFT and its distinct cell lineages, which is critical for understanding congenital heart defects and developing cardiac tissue for regenerative medicine.
In this study, we investigated how iron limitation alters central metabolism in <i>Mycobacterium tuberculosis</i> using metabolomics and stable isotope tracing. Our findings reveal a well-orchestrated metabolic programme to enable Krebs cycle activity despite the inefficient action of its iron-dependent enzymes. Under such conditions, carbon flux through the oxidative branch of the Krebs cycle is stalled, resulting in the accumulation of metabolites that are partially secreted. As a result, carbon flux from glycolysis is partially diverted to the reductive branch of the Krebs cycle to support the production of oxaloacetate and malate through the activity of phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Both branches terminate with the synthesis of malate, which is secreted. This unprecedented split of the Krebs cycle and malate secretion in a bacterial pathogen facilitates the continuous flow of carbon through the core of carbon metabolism, overcoming the metabolic stalling triggered by iron starvation.
In the low Reynolds number regime, active biological systems utilize nonreciprocal cyclic activities to achieve motility, as seen in the spinning of bacterial flagella and the beating of cilia. Coupling among these active mechanical components leads to synchronization and emergence of metachronal waves. Here, we report that biofilms of <i>Pseudomonas nitroreducens</i> form active carpet-like surfaces textured with diverse topological defects, generating Mexican-wave-like collective behavior in which bacteria periodically lift up. On these active surfaces, non-reciprocally coupled extension and retraction activities of bacterial pili drive these collective oscillations. Surprisingly, this collective behavior exhibits left-right asymmetry across the biofilm driving unidirectionally propagating waves. We discover that this directionality is primarily governed by an aging-related frequency gradient across the biofilm. Leveraging these insights, we further demonstrate the ability to control the collective dynamics of these waves, including symmetry breaking, transitions from spiral waves into target and propagating plane waves by manipulating the elastic properties of biofilms. Overall, our findings illuminate the fundamental role of nonreciprocally interacting active components in regulating synchronization, collective dynamics, and symmetry-breaking phenomena in biological systems.
Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present SynaptoTagMe, a suite of genetic tools in <i>Caenorhabditis elegans</i> to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the <i>C. elegans</i> nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.
We have developed a robust workflow to identify proline hydroxylation sites in proteins, combining hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-liquid chromatography-mass spectrometry (LC-MS) with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4993 and 3247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. Of these, 1954 (HEK293) and 1253 (RCC4) high-confidence non-collagen sites were inhibited by FG-4592. Hydroxylated peptides showed consistent characteristics across both datasets, including enrichment in more hydrophilic HILIC fractions and distinct charge and mass distributions compared to unmodified or oxidised peptides. The intensity of the diagnostic hydroxyproline immonium ion varied with MS collision energy, peptide concentration, and adjacent amino acid sequence. Using synthetic peptides, we demonstrate that combining LC retention time with optimised MS parameters enables reliable site identification, even with multiple proline residues present. Proteins with FG-4592-inhibited hydroxylation sites were enriched for roles in RNA metabolism, mRNA splicing, and cell cycle regulation, including the phosphatase 1 regulatory subunit Repo-Man (CDCA2).
PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression
Prolyl-hydroxylases (PHDs) are oxygen-sensing enzymes that mediate the hydroxylation of proline residues. In mammals, three PHD isoforms (PHD1–3) are responsible for proline hydroxylation of hypoxia-inducible factor (HIF) alpha, a key regulator of the hypoxia response. In the accompanying paper (Jiang et al., 2025), we report development of a mass spectrometry-based method to reliably identify proline hydroxylation (OH-Pro) sites on proteins and use this to identify a PHD-dependent OH-Pro modification at Pro604 on the protein RepoMan (CDCA2), a regulatory subunit for protein phosphatase PP1γ with important roles in mitotic progression and cell viability. Here, we investigate the functional significance of hydroxylation of RepoMan at P604. During M phase, the PP1-RepoMan complex dephosphorylates Thr3 of Histone H3 (H3T3) on chromosome arms to ensure the correct localisation of the chromosomal passenger complex (CPC) at centromeres. We show that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in prometaphase-arrested cells. In cells depleted of endogenous RepoMan, exogenous expression of wild-type RepoMan, but not a RepoMan-P604A mutant, restored normal H3T3 phosphorylation localisation in prometaphase arrested cells. RepoMan-P604 is located proximal to the short linear motifs (SLiMs) that function as binding sites for the serine/threonine protein phosphatase 2A (PP2A). The interaction of RepoMan and PP2A-B56γ is reduced in cells expressing RepoMan-P604A. Moreover, analyses in both fixed and live cells released from a prometaphase arrest show that expression of the RepoMan-P604A mutant delays completion of mitosis, results in defects in chromosome alignment and segregation, and increases levels of cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis, acting, at least in part, via hydroxylation of RepoMan at P604 regulating the interaction of RepoMan with PP2A during chromosome …
<p>by Teemu Kuosmanen, Juhani Rantanen, Dovydas Kičiatovas, Sanna Pausio, Ville-Petri Friman, Teppo Hiltunen, Ville Mustonen</p>
Understanding and predicting how communities assemble is a paramount challenge in ecology. Here we address these questions normatively by comparing the observed species abundance distribution to a game-theoretically fair distribution based on each species’ Shapley value. By analyzing in total 56 distinct community outcomes, we assess how fairly biomass is distributed in microbial communities displaying both competitive and cooperative interactions in different growth conditions. We find examples of fair communities that closely follow their Shapley value across all environments as well as counterexamples where the true abundances deviate from the species’ objective contribution to community biomass. Next, we develop a fair assembly rule based on the recursive definition of Shapley value and show that also unfair community compositions are consistent with the principles of fair assembly after the lower-level competitive outcomes are known. Our results give unique empirical insights into the distributive function of ecological dynamics and lay down the theoretical foundations of what might become a normative community assembly theory.
<p>by Han Gong, Kehui Liu, Shanjun Deng, Jinwen Wang, Xionglei He, Li Liu</p>
The dynamics of stem cell maintenance and proliferative patterns are key determinants of tissue aging in multicellular organisms. Leveraging our previously developed SMALT system with enhanced sequencing compatibility, we performed longitudinal lineage tracing of the adult <i>Drosophila melanogaster</i> midgut across different developmental stages. Using ubiquitous Tubulin-GAL4-driven labeling, we first profiled midgut-wide clonal dynamics during early adulthood (3–33 days post-eclosion). Phylogenetic reconstruction revealed that clonal diversity peaked immediately after eclosion and began to decline earlier than anticipated, accompanied by a reduction in effective population size. To further investigate stem cell-specific dynamics during late adulthood, we employed intestinal stem cell (ISC)-specific Dl-GAL4-driven labeling (33–63 days post-eclosion) and observed sustained clonal attrition in the posterior midgut. This progressive loss of diversity was consistent with an age-associated change in effective proliferative behavior and reduced lineage maintenance capacity, as reflected by a decline in net proliferative output inferred from lineage topology. Remarkably, ISC lineages emerging within the first 10 days post-eclosion exhibited sustained clonal dominance in aging populations, with a single lineage comprising over 63% of sampled cells by Day 63. Bayesian survival modeling confirmed that these early-origin lineages have the highest probabilities of long-term persistence, while a graph neural network model accurately predicted their structural evolution across successive stages. Together, we delineate a timeline for clonal attrition and deliver topology-driven predictors of clone survival and structural change, enabling prospective identification of dominant and failing clones during aging.
<p>by Ying Yao, Simon Hanslmayr</p>
A new study in PLOS Biology shows that neuronal firing is selectively tuned to oscillatory frequency in human intracranial recordings, complementary to phase tuning, suggesting an additional dimension in how brain rhythms may organize neural activity.
This primer discusses a study in PLOS Biology showing that neuronal firing is selectively tuned to oscillatory frequency in human intracranial recordings, complementary to phase tuning, and suggesting an additional dimension in how brain rhythms may organize neural activity.
Chen X-W, Wang H, Bajaj K, Zhang P, Meng Z-X, Ma D, Bai Y, Liu H-H, Adams E, Baines A, Yu G, Sartor MA, Zhang B, Yi Z, Lin J, Young SG, Schekman R, Ginsburg D. 2013. SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion. eLife 2:e00444. doi: 10.7554/eLife.00444.
Published 9 April 2013
We have been made aware through a notification from PubPeer that, in the above publication, the lung image in Figure 1 (the lower right section of panel F) appears to be a duplicate of an image in an earlier publication from our research group (lower right panel from Figure 5 in Thrombosis Research, 123:785–792, 2009, PMID: 18774162). The lung histologies in both animals (a wild-type control mouse in the 2009 paper and a Sec24agt/gt mouse in the 2013 eLife paper) were entirely normal, as determined by the expert pathologist who extensively reviewed the tissues in both animals, prepared both figures, and is a co-author on both papers. We have concluded that the error likely occurred during preparation of the eLife figure, at which time the earlier image of normal lung histology was accidentally inserted in place of an image of similar normal lung histology from the Sec24agt/gt mouse. We sincerely apologize for this error.
To correct this error in the eLife manuscript, panel F from Figure 1 has been removed, and the description of panel F in the Figure 1 legend has also been removed. Also, the text in the second paragraph of Results has been modified as follows:
Corrected text:
Gross and routine microscopic survey of multiple tissues failed to identify any obvious morphologic abnormalities in adult Sec24agt/gt mice.
Original text:
Gross and routine microscopic survey of multiple tissues failed to identify any obvious morphologic abnormalities in adult Sec24agt/gt mice (Figure 1F).
The corrected Figure 1 (with Panel F removed) is shown here:
Corrected Figure 1 legend text:
Figure 1. SEC24A null mice are viable and exhibit normal survival and development. (A) Schem…
Arousal fluctuates continuously during wakefulness, yet how these moment-to-moment variations shape large-scale functional connectivity (FC) remains unclear. Here, we combined 7T fMRI with concurrent pupillometry to quantify, for every functional connection, how time-varying FC covaries with spontaneous arousal in the awake human brain. Rather than exerting a uniform influence across the connectome, arousal organized FC into a low-dimensional set of seven connectivity communities, each defined by characteristic network compositions. These communities exhibited systematic hemispheric asymmetries, specifically identifying a ‘left-hemisphere centripetal architecture’ where the left hemisphere serves as a structural sink for the asymmetric convergence of arousal-modulated signals. Importantly, hemispheric asymmetry did not arise from global shifts in connectivity strength but instead reflected structured spatial heterogeneity embedded within community architecture. This modular and asymmetric organization was highly preserved during naturalistic movie watching, indicating that arousal-related modulation of FC reflects intrinsic principles that generalize across awake cognitive contexts. Together, these findings demonstrate that moment-to-moment arousal fluctuations shape large-scale FC through structured, hemispherically asymmetric network organization during wakefulness.
During the first period of life, human infants rapidly and effortlessly acquire the languages they are exposed to. Although memory is central to this process, the nature of early verbal memory systems, and the factors that determine retention and forgetting, remain largely unknown. Behavioral and brain measures have demonstrated memory formation in newborns. However, word traces fade in the face of acoustic overlap, leading to interference and forgetting. Here, we investigate whether speakers' identity changes facilitate the separation into distinct acoustic episodes and the creation of non-overlapping verbal memories. Newborns (0–4 days-old) were tested in a familiarization-interference-test protocol, while neural cortical activity was recorded using functional Near-Infrared Spectroscopy (fNIRS). The results showed higher neural activation to novel words than to familiar ones during the test phase, indicating that the infants recognized the familiar words despite potentially interfering sounds. The recognition response was measured over the left inferior frontal gyrus (IFG) and superior temporal gyrus (STG) areas known to be crucial for encoding auditory information and language processing. The neural response also included the right IFG and STG, involved in interpreting vocal social cues and speaker recognition. The results indicate that speaker identity is a key feature in the formation of verbal memories from birth, facilitating separability, possibly through early source–content binding (i.e. what–who), a precursor to fully mature episodic memory.
Tagging all proteins encoded by an animal genome with a fluorescent tag would open many windows to the discovery of unexpected patterns of protein expression and localization. To scale such an approach, it would be beneficial to introduce multiple, spectrally distinct fluorophore tags in parallel. As proof of concept for scalable pooled tagging, we undertook a pilot study in the nematode <i>Caenorhabditis elegans,</i> in which we set out to tag 30 different genetic loci with three different fluorophores, with three tags being introduced at a time. By choosing essential genes, predicted based on transcriptomics to cover a range of expression levels, we explore issues relating to disrupting gene function and visibility of tagged proteins. We demonstrate that such a tagging approach is highly efficient and indeed reveals unanticipated patterns of cellular and subcellular sites of protein expression and localization. We hope that this pilot study will motivate attempts to scale this tagging approach to more loci and, ultimately, the whole genome.
<p>by Marvin van Aalst, Alienor Lahlou, Tanvir Hassan, William Gaultier, David Colliaux, Anna Matuszyńska</p>
Web-based modeling platforms can enhance collaboration between modelers and experimentalists during early model development. Drawing on two interdisciplinary case studies, we provide guiding principles on how to build interactive agile modeling tools.
Web-based modelling platforms can enhance collaboration between modelers and experimentalists during early model development. This Community Page provides guiding principles on how to build agile interactive modelling tools.
<p>by Zahra Jourahmad, Raissa K. Mathura, Layth S. Mattar, Melissa C. Franch, Danika L. Paulo, Mohammed Hasen, Nicole R. Provenza, Benjamin Y. Hayden, Sameer A. Sheth, Eleonora Bartoli, Andrew J. Watrous</p>
Neural oscillations play a critical role in shaping neuronal firing patterns. While phase-locked neuronal firing (“phase tuning”) has been extensively studied in animal models and human invasive recordings, much less is known about whether neurons show preferential firing at specific oscillatory frequencies, termed frequency tuning. Here, we employ human intracranial recordings across several brain regions including hippocampus, entorhinal cortex, anterior and posterior cingulate cortex, and orbitofrontal cortex to test the hypothesis that neurons exhibit frequency-specific firing. We analyzed 357 single units recorded simultaneously with local field potentials in 19 neurosurgical patients during awake resting. We estimated the instantaneous frequency of the LFP using adaptive spectral decomposition and assessed frequency tuning of each neuron while controlling for changes in firing rate unrelated to frequency changes. We found 27% of neurons exhibited increased or decreased firing within specific frequencies, most commonly within the low-frequency range (<10 Hz). Neurons exhibiting frequency tuning were distinct from those displaying phase tuning, and both types of tuning were observed across multiple brain regions with no anatomical preference. Together, our results demonstrate that the instantaneous frequency of neural oscillations modulates neuronal firing which may serve as an additional mechanism for information processing in the human brain, opening new avenues for frequency-targeted neural stimulation.
<p>by David Elmenhorst, Anna L. Foerges, Ali Gordji-Nejad, Eva-Maria Elmenhorst, Tina Kroll, Andreas Matusch, Simone Beer, Bernd Neumaier, Philipp Krapf, Christoph Lerche, Alexander Drzezga, Andreas Bauer</p>
<p>Sleep is essential for synaptic homeostasis, a proposed mechanism whereby wakefulness leads to synaptic potentiation and sleep facilitates synaptic down-selection. Synaptic vesicle glycoprotein 2A (SV2A), whose availability is quantifiable by [¹⁸F]SynVesT-1 positron emission tomography (PET), is commonly interpreted as a proxy for synaptic density. In this randomized study, we examined 40 healthy adults (mean age 27.5 ± 6.5 years) who underwent two [¹⁸F]SynVesT-1 PET scans on consecutive days. Half of the participants were assigned to the normal sleep (i.e., control) condition and half to the sleep deprivation condition. Scans were performed at the same circadian time point, approximately 4 h after awakening in the control group and during baseline in the sleep deprivation group or after ~28 h of continuous wakefulness in the sleep deprivation group after sleep deprivation. Sleep deprivation led to significant increases in synaptic vesicle glycoprotein 2A binding in multiple brain regions, including the thalamus (+4.6%), hippocampus (+5.6%), and parietal cortex (+3.2%), whereas no changes were observed in controls. The degree of increase in synaptic vesicle glycoprotein 2A positively correlated with elevated slow wave activity during recovery sleep, a physiological marker of sleep pressure. These findings provide in vivo support for the synaptic homeostasis hypothesis in humans and suggest that synaptic vesicle glycoprotein 2A PET imaging is sensitive to sleep-wake dependent synaptic plasticity.</p> Trial Registration <p>The study was prospectively registered on 19.01.2022 here: German Clinical Trials Registry: DRKS # DRKS00027867, https://drks.de/search/en/trial/DRKS00027867.</p>
This Medical News article discusses ongoing developments in the Ebola outbreak primarily in the Democratic Republic of the Congo and the challenges in containing the rare Bundibugyo virus, which has no vaccine or specific treatment.
B. A. Thomas, M.D., Philadelphia
Among poetry’s applications in modern medicine is to reconnect clinicians to ancient healing rituals. In “After Sunstroke,” the speaker searingly recalls the Dionysian cult of ancient Greece and specifically its association with the maenads, female followers of the god of ecstasy who were believed to possess healing powers expressed in the catharsis of his worship with libation and dance. We hear the throb of music in the poem’s iambic rhythm, with the speaker also evoking the altered sensorium of sunstroke in a further reference to the madness of the maenads’ writhing and moaning. To the ancient Greeks, such rituals were community gatherings that themselves catalyzed healing; a similar impulse to (re)join in human communion is acutely felt here after the speaker recounts his isolating, hallucinatory symptoms of “I could glean/only goat bleat and the untuned bells/of a mad song…//…rereading the same/dense page about the German troops/in snow, till I would almost plead//to join them getting picked apart.” What feels like it could culminate in “certain death” is met instead by the healing physicality of the maenads, who seem to assuage not only the speaker’s, but even all humanity’s, suffering—that “piercing ring of heat”—in suffusing it with their “raised arms and wild hair.” Their frantic motion and headlong rush toward release is reminiscent, perhaps, of the hectic days of clinicians on the verge of burnout, always going beyond the possible in the quest to heal. Thus poetry aids in the timeless struggle to ease our patients’, and perhaps even our own, debasement by illness.
To the Editor Ms DeCleene and colleagues presented a methodologically robust analysis of obesity prevalence in the US from 1990 to 2022, with forecasts through 2035. Their analytic strategy leveraged modeling techniques from the Global Burden of Disease project, which has produced highly reliable estimates of disease burden in the US and globally. However, the current use of this approach for US obesity forecasting relies on the assumption that pre-2022 trends will continue, absent exogenous shocks. The rapid proliferation of glucagon-like peptide 1 receptor agonists (GLP-1 RAs) represents an already demonstrated example of such a shock.
To the Editor Dr Ali and colleagues reported no additional analgesic benefit from adding acetaminophen (paracetamol) or hydromorphone to ibuprofen for children with acute musculoskeletal injuries. In all groups, the ibuprofen dose was 10 mg/kg and the acetaminophen dose was 15 mg/kg, with pain assessed 60 minutes after drug administration. Pain scores did not differ between ibuprofen monotherapy and either combination (ibuprofen plus acetaminophen or ibuprofen plus hydromorphone).
To the Editor Dr Ali and colleagues reported results from 2 randomized clinical trials evaluating whether adding acetaminophen (paracetamol) or hydromorphone to ibuprofen improves analgesia for children with acute nonoperative musculoskeletal injury. In pooled analyses, pain scores at 60 minutes were similar across groups, whereas adverse events were more frequent with hydromorphone. These findings are timely given pediatric opioid stewardship policies and recent clinical guidance that encourages nonopioid-first strategies and cautious outpatient opioid prescribing for acute pain in children.
In Reply Drs Yin and Zhang query what impact clinically relevant heterogeneity of participants with severe pain scores might have on study conclusions for the No OUCH trials. Study exclusion criteria ensured children requiring intranasal or intravenous opioid analgesia for severe pain would not be included in this study of oral analgesia. Indeed, the study participants’ mean baseline pain severity score of 6.4 to 6.5 (SD, 1.7-1.9), which at its upper limits was still only at the cusp of severe pain (defined as 8-10/10), included the group in whom clinicians often consider escalation beyond ibuprofen. We agree with Yin and Zhang that all children with mild to moderately severe pain should receive oral analgesia. However, our study was specifically designed to understand the incremental benefit of adding other analgesics to ibuprofen for children with moderately severe pain, rather than those with severe pain. Stratification by injury type demonstrated no clinically or statistically significant difference between study groups and across injury types (fracture vs sprain vs dislocation) consistent with prior studies of children’s fractures and other musculoskeletal injuries that have not reported any benefit with either combination therapy or opioids alone. We plan to report functional outcomes according to injury type in a future article, in which we will consider stratification by pain severity if the data support such analysis.
To the Editor Dr Williams and colleagues reported a rapid recovery with extended ultraoxygenated preservation (REUP) technique for adult donation after circulatory death (DCD) heart transplant that avoids both preimplant donor heart reanimation and ex situ machine perfusion, with encouraging early outcomes (30-day survival, 96%; severe primary graft dysfunction, 4%). In settings where thoracoabdominal normothermic regional perfusion is restricted—often reflecting deceased donor rule concerns and variable institutional policies—a cold-preservation strategy that does not require reanimation could have important practical implications.