Dear Editor,1 Recent studies have shown that RNA modifications are likely to participate in the progression of HCC,2 suggesting new methods for tumor growth control. Specifically, 5-methylcytosine (m5C) is one of the most interesting RNA modification types, as accumulating evidence suggests its role in the promotion of HCC tumorigenesis.3 The RNA methyltransferase Aly/REF export factor (ALYREF) is considered one type of “reader” protein located in the nucleus that recognizes and binds directly with m5C sites in RNA and facilitates the export of RNA from the nucleus to the cytoplasm.4 Notably, ALYREF is considered a promising target for diagnosis and prognosis prediction.5 However, until now, the low number of related studies has limited the understanding of the mechanism of the HCC-promoting effects of ALYREF. To further elucidate the oncogenic roles of ALYREF in HCC, we assessed the expression levels of ALYREF in clinical samples and HCC cell lines and explored the effects of ALYREF deficiency by both in vitro and in vivo experiments and m5C-methylated RNA immunoprecipitation sequencing (m5C-MeRIP-Seq).The incidence of hepatocellular carcinoma (HCC) is increasing in many countries due to hepatic virus infection and unhealthy diet habits, and the unsatisfactory overall survival rate calls for novel treatments with higher efficacy.5C regulators, including NSUN1–7, DNMT1–2, DNMT3A, DNMT3B, ALYREF, and TET2, using RNA sequencing (RNA-seq)  Fig. . The ALYeq) Fig.  and quaneq) Fig. . As expeNext, the effect of ALYREF deficiency on Huh7 and HepG2 cells was investigated. Three types of short hairpin RNA (shRNA) were used for ALYREF knockdown, and knockdown efficiency was validated by Western blotting Fig. , qRT-PCR5C modification, we then performed m5C-MeRIP-Seq on both sh-NC and sh-ALYREF Huh7 and HepG2 cells. A total of 109,263 and 109,277 m5C sites of mRNA were found in sh-NC and sh-ALYREF Huh7 cells, respectively, and the two groups shared a majority of m5C-methylated mRNA sites  to identify target genes that might bind to ALYREF in Huh7 cells. The results showed that a total of 295 peaks were targeted by ALYREF from IP samples based on three independent replicates Fig. . The proein Fig. . Taken t5C-targets needs to be fully characterized in the further studies. Hopefully, our study will provide novel insight into ALYREF-based targeted therapy for HCC.In summary, our results increase the understanding of the role that ALYREF plays in HCC from a novel and broader perspective. The abundance of ALYREF in tumor tissues and HCC cell lines, along with the tumor-suppressive effects of ALYREF deficiency in vitro and in vivo, confirmed the oncogenic roles of ALYREF in HCC development. Mechanistically, our study suggests that ALYREF promotes HCC by regulating methylation levels of target genes. However, the axis of ALYREF-mSUPPLEMENTAL MATERIAL


Decision-making forms a central bottleneck to most of our tasks, one that people often experience as costly. To mitigate these costs, previous work has proposed adjusting one’s threshold for deciding  to avoid over-deliberating. Here, we test an alternative solution to these costs, one that targets the basis for most choice costs: the fact that choosing one option sacrifices others . Across 4 studies (N = 385), we test whether this tension can be relieved by framing choices as inclusive , and whether doing so improves decision-making and the experience thereof. We find that inclusivity makes choices more efficient, because of its unique impact on the level of competition between potential responses as participants accumulate information for each of their options (resulting in a more “race”-like decision process). We find that inclusivity also reduces the subjective costs associated with choice, making people feel less conflicted in conditions where it was hard to choose which good option to acquire or which bad option to get rid of. These inclusivity benefits were distinct from those achieved when trying to merely reduce deliberation , which we show can in some cases lead to similar increases in efficiency but only carry the potential to diminish not improve the experience of choosing. This work collectively provides key mechanistic insights into the conditions under which decision making is most costly, and a novel approach aimed at mitigating those costs. Humans are capable of making remarkably complex decisions, integrating over a multitude of factors and timescales , and yetmutually exclusive of one another . This mutual exclusivity creates a tension whereby a person feels a tug towards and against each of their options  versus accuracy (choosing the best option in a set). For instance, rather than trying to select the best possible option, a person can choose the first option that meets a certain set of criteria  and they can guarantee that a choice is ultimately made. However, lower thresholds also necessarily come with the cost of a potential sacrifice to choice accuracy . Moreoveexclusive choice) and under conditions where they can go on to select other options from that set (inclusive choice). Despite this added flexibility, we show that participants still choose their favorite option first in the inclusive condition, and they do so more efficiently than in the exclusive condition. We show that these and other patterns of choice behavior from our experiment are selectively accounted for by a computational model in which choice inclusivity reduces the level of competition  between potential responses. We further show that manipulations of choice inclusivity generate distinct behavioral patterns from, and confer unique benefits relative to, changes in choice urgency resulting from tighter deadlines. Most notably, unlike urgent choices, inclusive choices feel less conflicting than their alternative . Tying this work to recent studies identifying the conditions under which choice costs are greatest , this was the only choice participants made from the set; for the other half of trials (inclusive choices), participants were allowed to subsequently choose as many additional options from the set as they preferred. Exclusive and inclusive choices were interleaved throughout the session and were explicitly cued by a colored fixation cross prior to and throughout the trial. Critically, irrespective of the choice type, participants were always told to select the best item first. Choice sets were constructed to vary in the overall and relative value of the options, based on item-wise ratings given by participants earlier in the session. After making all of the choices, participants viewed each choice set again and retrospectively rated the level of choice conflict they had experienced while engaging in that choice  for each condition by dividing the value of the chosen item by the time taken to make a given response, confining to the initial choice on each trial. We found that participants achieved a significantly higher reward rate for these initial choices when choices were inclusive  value of the choice set  or especially low in value (inducing high levels of conflict over which option was least unpreferred) , they felt less conflicted and chose more efficiently. Interestingly, choice inclusivity led to reduced choice conflict for most choices, but not when choosing between the least valuable options. This pattern is consistent with previous work suggesting that conflict for low-value choices stems from not wanting any of the options  as partiselect, we instead had participants assume a set of options had already been selected for them and asked them to choose which option(s) they wanted to de-select  from that set  but not when choosing which one to remove (Study 2). Conversely, when deciding between low-value options, relaxing choice exclusivity diminished their experience of choice conflict when choosing which one to remove (Study 2) but not when choosing which one to select (Study 1).Both of these predictions were confirmed. First, just as participants choosing which option they most wanted from a set (Study 1), participants choosing which item they most wanted to remove from a set were faster  and de-select options that they would like to remove (Study 2). One potential concern, though, is that participants in both studies were given a limited time window to respond (9 s) which, while reasonably long for a single choice, may have introduced additional time pressure when participants were able to select up to four options (inclusive choices). Previous work suggests that such time pressure can result in a dynamically decreasing response threshold  which coWe followed the same analysis procedure as in Study 1 and 2, except that to account for the long tail in RTs without the time limit, we log-transformed RTs prior to the analysis. Our results confirm our previous findings. Across both tasks, participants responded faster for inclusive relative to exclusive choices  or low urgency .Previous work suggests that decisions can be optimized by tightening one’s decision deadline, constraining their natural inclination towards setting their decision thresholds too high  and less accurate . Consistent with previous demonstrations of urgency’s utility for choice optimization . Instead, urgency seems to undercut one of the features of choice sets that typically promotes lower choice conflict: relative value. Across both exclusive and inclusive choices in Study 1 and our low-urgency exclusive choices in Study 4, we found that people experience less conflict the higher the relative value of their choice set , consistent with past findings  and removed fewer options  the more valuable those options  and removal , these choices afford us a unique opportunity to understand how people evaluate options under conditions where choice is voluntary. In particular, we could examine how people choose how many items to (de-)select, and how this was related to experiences of choice conflict towards the initial set of options. We found that these voluntary choices were heavily determined by the overall value of the choice set, such that participants selected more options . To examine whether keeping these additional options on high-conflict trials reflected more or less optimal decision-making, we counted the number of options that were kept on a given trial despite having a value lower than the within-subject mean value of all possible options in the study. We found that higher levels of experienced choice conflict at the start of a given choice set predicted keeping a higher proportion of these sub-par options . We found that experiencing the initial choice as more conflicting led participants to select more options in Studies 1 and 3A  or inclusive . We found that participants were more efficient and felt less conflicted when making inclusive choices, independent of the choice goal  and in both the presence and absence of time pressure. We showed that the patterns of choice behavior we observed when participants were making inclusive relative to exclusive choices – including a selective enhancement of the influence of overall value on RT without altering the influence of other value estimates on behavior – was uniquely accounted for by a model in which choice inclusivity resulted in a relaxation of mutual inhibition between the competing options. These patterns of behavior and choice conflict were distinct from those resulting from a change in response deadline.Previous work has shown that parameters of the decision process are sensitive to one’s environment and current goals, for instance, their rate of accumulating evidence and their threshold for making a decision . This isReplicating our previous studies of exclusive choice , we showγ-aminobutyric acid, an inhibitory neurotransmitter; Decision-making dysfunctions are common across a wide range of psychiatric disorders , such asOur findings are also applicable to optimizing a wide array of real-world decisions. While we cannot turn most of our choices into buffets in the sense of being able to have all of our options at the same time, many real-world choices are actually less terminal than we may treat them - purchased products can typically be returned, written words can often be revised, and most other choices can be revisited in some fashion. In other words, choices may be exclusive in the moment but inclusive over time. It is thus tempting to consider the forms of choice exclusivity studied in our experiments as falling on a broader spectrum of choice behaviors that have been studied under the rubric of deferral  and/or rimpair choice by necessarily trading off speed for accuracy. However, even setting aside concerns about choice performance, reducing one’s decision threshold  can also have consequences for experiences of negative affect  during and after choice. Within our own data, we see for instance that shorter deadlines lead participants to feel similar levels of conflict even when choices are particularly easy, potentially because of the greater potential for making an error on these trials.Our work also shows that choice inclusivity affords unique benefits relative to a prominent alternative strategy for choice optimization, which is to lower one’s threshold for deciding. Whereas decisions are predicted to only improve  with increasing inclusivity, threshold reductions can only benefit choice within a limited range, by bringing thresholds down from being overly conservative (overly cautious) to their optimal levels. Critically, past that point, threshold reductions Our computational and empirical findings point to a deeper puzzle: if mutual inhibition is maladaptive for optimizing decisions and experiences thereof, what benefit does it afford? Before examining this further, it is important to note that mutual inhibition’s role in choice does not appear not to be universal in the animal world. For instance, starlings make value-based decisions in a manner that resembles a race process  and choice set values for which choice inclusivity can prove most beneficial, and which can collectively point to conditions that are most conducive to efficient decision making and which can, conversely, prevent significant bottlenecks in everyday tasks.Across all four studies, participants (N=385) received monetary compensation, and provided informed consent in a manner approved by Brown University’s Institutional Review Board under protocol 1606001529.SDvalue < 1) or too high ; (2) to ensure compliance with the task instructions, we calculated participants’ choice consistency within the easy trials , and excluded participants whose mean accuracy in easy trials was less than 25%; (3) we also excluded participants with too low variance in their conflict ratings (SDconflict < 0.5). This resulted in a sample of 65 participants for Study 1B  and no exclusions for Study 1A. The qualitative patterns reported in this paper hold when we include all 74 Study 1B participants.17 participants  participated in Study 1A (in-lab), and 74  participants were recruited for Study 1B, an online replication study on Prolific. Participants were excluded from our analysis based on the following criteria: (1) to ensure that participants’ product ratings prior to the choice task cover the full range of the liking scale, we excluded participants whose standard deviation of their product ratings was too low . In both conditions, participants were given up to 9s to complete each trial and, importantly, were instructed to always start by selecting their favorite option out of the set. In Phase 3, participants viewed each choice set again and rated the level of conflict they felt when facing each set on a 5-point scale.Our experiment consisted of three phases . In PhasFor the choice phase, we used linear mixed effect regressions  of the overall (mean) value of the choice set, the relative value , the interactions between choice inclusivity and overall/relative values, and trial order, with random (subject-specific) intercept and slopes for each variable. We also tested additional models with control of reaction time and choice accuracy see .118 participants  participated were recruited for this study on Prolific. Participants were excluded from our analysis based on the same criteria for Study 1. This resulted in a sample of 98 participants for Study 2 . The qualitative patterns reported in this paper hold when we include all 118 participants.exclusive choice trials, participants were allowed to deselect one product from the choice set. Once they clicked on this product, the box around it disappeared and they proceeded to the next trial. On inclusive choice trials, participants were able to continue deselecting as many options as they preferred after they deselected the first one. The two choice conditions were intermixed, occurred with equal likelihood, and were cued by the color of the fixation cross in the middle of the screen (blue for exclusive deselections and green for inclusive deselections). In both conditions, participants were given up to 9s to complete each trial and, importantly, were instructed to always start by deselecting their least favorite option out of the set. In Phase 3, participants viewed each choice set again and rated the amount of conflict they felt when facing each set on a 5-point scale.The procedure of the experiment is the same with Study 1, except that participants were instructed to make deselections among sets of four pre-selected products framed with boxes. On For the choice phase, we analyzed reaction time (RT) and choice accuracy (whether the lowest-rated option was deselected) for the first choice in each condition. The setup of predictors is the same with Study 1, except for the relative value .68 participants  participated in the selection task (Study 3A), and 78  participants were recruited for the deselection task (Study 3B). Participants were excluded from our analysis based on the same criteria for Study 1 and 2. This resulted in a sample of 59 participants for selection task  and 61  for the deselection task. The qualitative patterns reported in this paper hold when we include all participants.The replication studies follow the same procedures of selection (Study 1) and deselection study (Study 2) with the variance that 1) we removed the choice deadline and 2) we selected a new set of products (N=210).We followed the same analysis settings in Study 1-2. The only difference is that we log-transformed the reaction time to account for the long-tail distribution.108 participants  participated were recruited for this study on Prolific. In addition to the exclusion criteria for Study 1, participants with high omission rate in high urgency choice trials (>=40%) were also excluded from our analysis. This resulted in a sample of 85 participants for Study 4 . The qualitative patterns reported in this paper hold when we include all 108 participants.exclusive selections with low or high urgency. On low urgency choice trials, participants have unlimited time to make their choice. On high urgency choice trials, participants have only 3 seconds to make their choice. The two choice conditions were intermixed, occurred with equal likelihood, and were cued by the color of the fixation cross in the middle of the screen . In Phase 3, participants viewed each choice set again and rated the amount of conflict they felt when facing each set on a 5-point scale.The procedure of the experiment is the same with Study 1, except that participants were instructed to make The setup of analysis is the same with Study 1, except now low and high urgency are coded as contrast in the model.(a) number of selections/removals and (b) number of products kept for each trial . We then examined the quality of subsequent choices. We analyzed (c) probability of selection/removal per product and (d) likelihood of keeping suboptimal products  per trial. For (a), (b) and (d), the model included the linear and quadratic terms  of the overall (mean) value of the choice set, the relative value , the reaction time and accuracy of initial choices, and trial order. For (c), the model included overall value and the difference between product value and subject-specific global average. For (b) and (d), we also performed the same analysis after combining selection and removal studies. All of these models included random (subject-specific) intercept and slopes for each variable.We only included inclusive choice in this analysis and combined studies based on type of choice . We analyzed the number of inclusive choices in each trial by fitting linear mixed models to Supplement 1


Regulation of RNA cap formation has potent impacts on gene regulation, controlling which transcripts are expressed, processed and translated into protein. Recently, the RNA cap methyltransferases RNA guanine-7 methyltransferase (RNMT) and cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (CMTR1) have been found to be independently regulated during embryonic stem (ES) cell differentiation controlling the expression of overlapping and distinct protein families. During neural differentiation, RNMT is repressed and CMTR1 is up-regulated. RNMT promotes expression of the pluripotency-associated gene products; repression of the RNMT complex (RNMT–RAM) is required for repression of these RNAs and proteins during differentiation. The predominant RNA targets of CMTR1 encode the histones and ribosomal proteins (RPs). CMTR1 up-regulation is required to maintain the expression of histones and RPs during differentiation and to maintain DNA replication, RNA translation and cell proliferation. Thus the co-ordinate regulation of RNMT and CMTR1 is required for different aspects of ES cell differentiation. In this review, we discuss the mechanisms by which RNMT and CMTR1 are independently regulated during ES cell differentiation and explore how this influences the co-ordinated gene regulation required of emerging cell lineages. Uncovering the molecular mechanisms operating in embryonic stem (ES) cells is critical for our understanding of development and many disease states, and for the use of these cells in therapeutics. The function and fate of ES cells are governed by networks of transcription and translation which are co-ordinately controlled during differentiation programmes ,2. As inThe mammalian RNA cap is formed by a series of enzymes ,4,25 Fi. During Methylation of the cap nucleotides by RNMT, CMTR1 and CAPAM occurs predominantly co-transcriptionally when the enzymes are recruited to the nascent cap, RNA and/or RNA pol II . CMTR2 mIn ES cells, the pluripotent state is maintained by open chromatin and transcriptional plasticity . During In ES cells, the transcripts whose expression is most dependent on RNMT and CMTR1 were analysed by RNA sequencing ,24. It iHow do RNMT–RAM and CMTR1 regulate different RNAs ,24? AlthSince RNMT binds to nascent RNA as it is being transcribed by RNA pol II , and CMTSaccharomyces cerevisiae homologue of RNMT, ABD1 . Ex. Ex23]. Histone genes and ribosomal protein (RP) genes transcripts are the gene families most dependent on CMTR1 in ES cells . However, which can be observed in the metadata analysis and in the analysis of individual RNAs and proteins in ESCs, including during differentiation (As discussed, the repression of RNMT–RAM results in the repression of many pluripotency-associated gene transcripts and proteinsntiation  23]. A . A , whintiation  24].., which The histones are the predominant CMTR1 target gene transcript family . Here, wTo conclude, the cap methyltransferases RNMT and CMTR1 control the expression of distinct sets of gene products. By examining their mechanisms of action and how they are recruited to their substrates we can begin to understand why their target genes differ. During ES cell differentiation, the independent regulation of RNMT and CMTR1 facilitates the co-ordinate regulation of the pluripotency-associated genes, histones and RPs required for differentiation and proliferation during this process.Major cellular events are accompanied by regulation of the RNA capping enzymes, which can direct co-ordinated and coherent regulation of gene expression and influence cell proliferation, cell differentiation and immune responses. Experimentally we can direct capping enzyme functionality and therefore development of therapeutic strategies to modify capping enzyme expression or catalytic activity is possible. Such cap therapeutics may have uses in regenerative medicine, cancer therapeutics and the treatment of neurological disorders, when directed interference in gene regulation has value.Regulation of the RNA cap methyltransferases impacts on many steps in gene expression and indirect impacts on cell functions and cell fate decisions.Here, we discuss how RNA cap methyltransferase regulation during ES cell differentiation impacts on RNA expression associated with pluripotency, translation, DNA replication and proliferation.Gene expression in other stem cells is likely to be influenced by the regulation of RNA cap methylation, with the specific genes regulated being tissue dependent.


Control over the electrical contact to an individual molecule is one of the biggest challenges in molecular optoelectronics. The mounting of individual chromophores on extended tripodal scaffolds enables both efficient electrical and mechanical decoupling of individual chromophores from metallic leads. Core-substituted naphthalene diimides fixed perpendicular to a gold substrate by a covalently attached extended tripod display high stability with well-defined and efficient electroluminescence down to the single-molecule level. The molecularly controlled spatial arrangement balances the electric conduction for electroluminescence and the insulation to avoid non-radiative carrier recombination, enabling the spectrally and spatially resolved electroluminescence of individual self-decoupled chromophores in a scanning tunneling microscope. Hot luminescence bands are even visible in single self-decoupled chromophores, documenting the mechanical decoupling between the vibrons of the chromophore and the substrate. A fundamental challenge for molecular electronics is the change in photophysical properties of molecules upon direct electrical contact. Here, the authors observe hot luminescence emitted by single-molecule chromophores that are electrically and mechanically self-decoupled by a tripodal scaffold. When single molecules are to be used as electrically powered photon sources, their coupling to the electrodes has to be optimized: Light emission from the chromophore is hampered when their orbitals are too strongly hybridized with the metallic leads due to quenching of the chromophore’s excited state by the metal’s free electrons, but it is not functional either when electrically isolated so that no current passes through. From an experimental view-point, the vacuum gap of a scanning tunneling microscope (STM) provides a tuneable electric contact to single molecules6. However, the coupling to the second lead, provided by the metal substrate, has to be reduced, and this is typically achieved by a thin insulating layer with a large band gap between the metal surface and the chromophores16. A purely molecular approach of integrating the insulating spacer between chromophore and substrate by molecular design is less explored in spite of its conceptual attractiveness and application potential. Particularly appealing are multipodal platforms mounting the optically active molecular subunit not only at a well-defined distance above the metal surface, but also controlling its spatial arrangement18. A variety of molecular architectures combining footing platforms with exposed chromophores have been developed and integrated in STM-based electroluminescence experiments24. The diversity of observed emission features in the individual experiments and/or the limited stability of the emitting junctions hint at the challenges that remain in competition with experimental set-ups that rely on insulating layers.Control of the molecular arrangement and spatial orientation of light-emitting molecules on metallic surfaces is of crucial importance for the construction of artificial photonic devices such as organic light-emitting diodes or single photon sources for quantum optical technologies18. The strength of the platform immobilization on the substrate depends on the nature of the interaction between them. Flat delocalized molecular subunits tend to interact with the planar substrate by physisorption with a moderate binding energy enabling lateral self-assembly to periodically arrange molecules in monolayers, but reducing the probability of separated individual molecules required for single molecule studies19. An appealing alternative is the decoration of the platform with thiol anchor groups forming covalent sulfur-gold bonds with the substrate25. The thereby chemisorbed system has a substantially increased binding energy enlarging the probability for separated single molecules. The lateral dimension of the platform turns out to be of crucial importance. In a first attempt with a tripodal platform of smaller dimension, the physisorption of the mounted chromophore was favored over the chemisorption of the foot structure, resulting in a flat arrangement of the chromophore on the surface, additionally stabilized by two of the three anchor groups forming covalent bonds24. An effective strategy to increase the interaction of the foot structure with the substrate is to enlarge its size26, with the additional advantage that the distance between perpendicularly mounted chromophore is increased, avoiding quenching by inter-chromophore stacking. Finally, structural rigidity of the platform is required to obtain control over the spatial arrangement of the emitting chromophore. With a central sp3-hybridized carbon atom as a mounting point, enlarging the π-systems of the three footing substituents increases their chemisorption and thereby planarizes the entire foot structure and forces the fourth substituent into a perpendicular arrangement. The crucial parameter of the electroluminescence experiment, the extent of coupling between the chromophore mounted as the fourth substituent and the substrate, becomes adjustable by its linking structure. The interaction between the mounted chromophore and the substrate depends on the chemical nature of the linking structure, the distance and orientation, as the transition dipole moment of the chromophore should have substantial contributions perpendicular to the surface to favor strong plasmonic enhancement29.A key to a spatially well-defined arrangement of the mounted chromophore is the molecular design of the platform scaffold. Numerous parameters have to be optimized like (i) the strength of the interaction between platform and substrate, (ii) the lateral dimension of the platform and (iii) its structural rigidity and integrityTol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac, with s/n indicating the heteroatom of the core substituent) were developed and synthesized for the experiment combining a 2,6-disubstituted naphthalene-1,4,5,8-tetracarboxdiimide (NDI) chromophore with a tripodal foot structure to control both their immobilization on and their interaction with the Au (111) surface. The tripodal platform Tol-Tpd with its thiol decorated tolane π-systems fixes the model compound firmly on the metal substrate and ensures an upright arrangement of the mounted chromophore. The phenylethynyl linker between the foot structure and the chromophore decouples the latter from the substrate. The model compounds were introduced into the ultrahigh vacuum (UHV) STM experiment with an in-house developed spray technique, which resulted in separated individual molecules, Tol-Tpd-sNDI or Tol-Tpd-nNDI, standing upright on their foot structures. Here, the low temperature STM based electroluminescence investigations show that the optical properties of the NDI chromophore at the single molecule level are preserved, including not only the tuneability by core substitution, but also resolved vibrational features and relatively high luminescence quantum yields. The integrity and stability of the immobilized molecule enables systematic STM-induced luminescence (STML) experiments including photon maps, which so far were only possible for molecules decoupled via insulating layers. In addition to the electrical decoupling, the tripodal scaffold also provides an impressive degree of mechanical decoupling of the chromophore, resulting in hot luminescence due to the increased lifetimes of the chromophores’ vibrational modes. This chemical approach to self-decoupling is not limited to the presently reported NDI dyes, its modular assembly allows to alter the exposed chromophore to further optimize light emitting properties of the molecular junction. Furthermore, the structural integrity and rigidity of the immobilized platform on the surface provides control over the orientation of the chromophore and thus also of its transition dipole moment.In this paper, we report single-molecule STM electroluminescence experiments with self-decoupled chromophores. Two model compounds methane23, the physisorption of the chromophore efficiently competed with the intended fixation by the three foot subunits of the tripod, pointing at the subtle balance between the surface interactions involved23. As the size of the chromophore in the presently reported second series remains the same, the foot structure was enlarged by three tolane subunits, increasing its lateral dimension and thus also its interaction with the surface. The strategy to increase the likelihood of an upright orientation by enlarging the footprint of the tripod was already reported by Yao and Tour26. The combination of the three physisorbed tolane π-systems with the chemisorbed thiol groups should firmly fix the foot structure to the surface and thereby ensure an upright orientation of the fourth substituent. The NDI chromophores are mounted through the rigid ethynylphenyl spacer, lifting the chromophore above the surface and decoupling it electronically, which also leads to a pronounced mechanical decoupling, at least in the low temperature STM experiment. After these considerations, the proposed tripodal chromophores are based on an extended tripodal architecture with three acetyl‐protected thiol anchors in the meta positions of the three tolane arms and NDI chromophores with 2,4,6-trimethylphenylsulfanyl (sNDI) and pyrrolidine (nNDI) core-substitution. The final steps of the assembly of the target molecules Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac are outlined in Fig. Tol-Tpd-H and both core-substituted NDI chromophores (see Methods and Supplementary Methods), Sonogashira reaction was used to couple sNDI-I and nNDI-I molecules with the modular platform Tol-Tpd-H to afford precursors Tol-Tpd-sNDI-TMSE and Tol-Tpd-nNDI-TMSE. Final transprotection of the thiols was successfully performed using AgBF4 and acetyl chloride, yielding the desired acetyl masked target structures Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac. Acetyl protecting groups, which prevent the accidental formation of oligo- and/or polymeric species by the formation of disulfides, e.g. by molecular oxygen, turned out to be particularly useful and, furthermore, can be easily deprotected either in situ upon binding to the gold surface or chemically by cleaving agents.Naphthalene diimides are compact, planar, and stable chromophores with electronic and optical properties that can be adjusted by the core substituentsTol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac were deposited as dichloromethane solution onto an atomically clean Au(111) surface by an in-house developed spray technique, which was optimized and successfully applied for tripod model compounds which cannot be sublimed, since their degradation temperatures lie below their sublimation temperatures33. Subsequent thermal annealing before cooling down to measurement temperatures promotes deprotection of the thiol anchors and the intended upright configuration with the chromophore pointing away from the substrate (details of the sample preparation can be found in Methods). Figure Tol-Tpd-sNDI and Tol-Tpd-nNDI junctions with the tripodal scaffold anchored to the Au(111) substrate, keeping the chromophores (nNDI and sNDI) away from the metal surface. Typical STM images of such samples . The calculated absorption spectra, corrected for the adiabatic energy shift, are shown in Suppl. Fig. z-axis  is fixed  is three orders of magnitude lower than those of Tol-Tpd-nNDI junctions, which is consistent with the relative fluorescence quantum yields observed for the corresponding chromophores in solution23 and our gas phase calculations . For both Tol-Tpd-nNDI and Tol-Tpd-sNDI emission of Qn and Qs is observed as soon as the energy of the tunneling electrons equals the energy of the emission lines Qn and Qs. Based on previous studies, this onset of STML, once the corresponding excitation energy is provided by the tunneling electrons, is an indication that the chromophore is excited by inelastic energy transfer in the plasmonic junction39. This is in full agreement with STML experiments at different tunneling currents  on both molecules  are observed at −2 V and −2.4 V for Tol-Tpd-sNDI and Tol-Tpd-nNDI, respectively , excluding excitation via sequential charging and discharging of the molecule at voltages up to 2.36 V. The onset of an additional excitation mechanism via sequential charging and discharging of the molecule would result in a step-wise increase of the photon emission rate12, which we do not observe for voltages up to 3 V . Simultaneously, STML spectra are recorded at each pixel and the integrated photon count of the Qn line is plotted  , which suggests that the same vibrational modes are involved. In full agreement with the literature42, the above observations can be explained in the following way: The main line results from transitions from the first electronically excited state (S1) to the electronic ground state (S0) without a change in the vibrational state  molecules on NaCl46. HL occurs when the time of the photonic de-excitation is similar or shorter compared to vibrational de-excitation, which is typically not the case (Kasha’s rule). There are two possible scenarios for this to happen: (1) The photonic de-excitation is drastically accelerated by coupling to gap plasmon-polaritons47. (2) The vibrational de-excitation is suppressed due to mechanical decoupling. Scenario 1, which has been used to rationalize previous observations of HL47, is an effect of low energy selectivity, as is reflected in the broad gap-plasmon spectrum  with a significant contribution of the foot structure , involving the corresponding FCF00². The lines on the LE side also involve excitation without change of the vibrational quantum number (FCF00), but are followed by an electronic de-excitation to a vibrationally excited state (FCF01). Thus, the ratio of the intensities of the two lines Qn /LE is given by (FCF00)²/(FCF00 × FCF01) = FCF00/FCF01. For the lines on the HL side, both the electronic excitation and de-excitation involve a change in the vibrational quantum number, i.e., FCF01 and FCF10, which can be approximated to be identical. With this, also the intensity ratio of LE/HE = (FCF00 × FCF01)/(FCF10 × FCF01) ≈ FCF00/FCF01. We tested this identity for the C = O line and show both ratios as a function of voltage and current in Fig. The STML spectrum recorded at see Fig. , so it csee Fig. . In ordeThe combination of tailor-made model compounds and high-end STM-based luminescence experiment provides improved light emission with high spectral resolution, intensity and stability for self-decoupled molecules, providing a route for efficient single molecular light sources. Crucial for this approach is that the foot structure provides a rigid anchor to the substrate that overcomes van der Waals attraction of the chromophore to the substrate providing upright oriented compact NDI chromophores upon spray deposition onto the sample and subsequent annealing. Further, this process offers the possibility of upscaling with the ability to orient the transition dipoles for maximizing the quantum yield and orienting push-pull complexes in the right direction within an OLED structure. It might help to overcome the limitations of OLEDs due to their disordered structure.−3 photons per electron and is robust enough to allow for detailed STML experiments, including photon maps of an individual molecule. Furthermore, the chromophore mounted on the tripodal scaffold is also mechanically decoupled, increasing the lifetime of vibrational excited states indicated by hot luminescence bands. In principle, the approach with even extended linkers could permit the study of chromophores with much longer recombination lifetimes making single molecules available as highly efficient single photon sources of narrow band width.The single molecule junction has a very high efficiency with up to 2.7 × 10The reported experiments document the potential of the tripodal foot structure and we are currently exploring its potential and limitation for the spatially controlled immobilization of functional subunits, ranging from optically addressed chromophores to molecular machinery.Tol-Tpd-H ethynylmagnesium bromide afforded trimethylsilyl (TMS) protected 3,3,3-tris(4-bromophenyl)propyne scaffold 3 in 78% yield, which is suitable for the further functionalization through Sonogashira cross-coupling reactions with the corresponding phenylacetylene 5. The thiol group of 3-bromothiophenol was protected as 2-(trimethylsilyl)ethyl derivative according published procedure with trimethyl(vinyl)silane through a radical reaction in the presence of 2,2′-azobis(2-methylpropionitrile) (AIBN) as a radical initiator to provide 2-(trimethylsilyl)ethyl (TMSE) derivative 4 in 93% yield32. The following Sonogashira reaction of bromo derivative 4 with trimethylsilylacetylene and the subsequent cleavage of the TMS protecting group led to the meta-substituted phenylacetylene 5 with high yield. This phenylacetylene was coupled further with the previously prepared 3,3,3-tris(4-bromophenyl)propyne scaffold 3 via Sonogashira protocol to afford an extended platform Tol-Tpd-TMS in 77% yield. Final deprotection of TMS group yield the desired extended platform Tol-Tpd-H in 97%, which can be modularly functionalized in the perpendicular position.The synthesis of an extended platform d-H Fig.  started Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac is outlined in Fig. sNDI-I derivative23 on the tolane based tripodal platform Tol-Tpd-H in 74% yield. Due to the poor solubility of NDI derivatives in neat amines, THF was used as a co-solvent. The subsequent transprotection of TMSE protected thiolate Tol-Tpd-sNDI-TMSE afforded the desired target molecule Tol-Tpd-sNDI-Ac in 62% yield. Similar to compound Tol-Tpd-sNDI-TMSE, we mounted nNDI-I derivative23 on the tolane based tripodal platform Tol-Tpd-H in 71% yield. The transprotection of Tol-Tpd-nNDI-TMSE to the corresponding thioacetate afforded the target molecule Tol-Tpd-nNDI-Ac in 76% yield. Both tripodal chromophores Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac bearing three thiacetate anchoring groups at meta-positions relative to the ethynyl groups, which are easily cleaved in situ during their deposition, provided successful coupling to the metal surfaces via three sulfur bonds.Final assembly of tripodal chromophores 2 under nitrogen atmosphere. TLC was performed on silica gel 60 F254 plates, spots were detected by fluorescence quenching under UV light at 254 and 366 nm. Column chromatography was performed on silica gel 60 (particle size 0.040–0.063 mm). Compound 432, sNDI-I, and nNDI-I23 were prepared according to a published procedure. Detailed synthetic procedures for all previously unpublished compounds and their full characterization are provided in Supplementary Information. All compounds were purified by chromatography and fully characterized by means of conventional NMR, FTIR, and UV–vis spectroscopy, mass spectroscopy, as well as by elemental analysis.All starting materials and reagents were purchased from commercial suppliers Alfa Aesar , Sigma-Aldrich , TCI Chemicals Europe , Merck  and used without further purification. The purity of all commercially available chemicals used was higher than 98%. Solvents utilized for crystallization, chromatography and extraction were used in technical grade. Anhydrous tetrahydrofuran and dichloromethane were taken from MBraun Solvent Purification System equipped with drying columns. Triethylamine was dried and distilled from CaH3. 1H NMR (500 MHz) spectra were referred to the solvent residual proton signal . 13C NMR (126 MHz) with total decoupling of protons were referred to the solvent . For correct assignment of both 1H and 13C NMR spectra, 1H-1H COSY, 13C DEPT-135, HSQC, and HMBC experiments were performed. UV–vis absorption spectra were recorded with an Agilent Cary 5000 spectrophotometer in a 1 cm quartz cell at ambient temperature in dichloromethane. Fluorescence PL spectra were measured with a Varian Cary Eclipse Fluorescence spectrometer at room temperature in a 1 cm quartz cell in dichloromethane. The molecule Tol-Tpd-sNDI-Ac was excited at 370 nm and both excitation and emission resolution were set to 20 nm. The molecule Tol-Tpd-nNDI-Ac was excited at 366 nm and both excitation and emission resolution were set to 10 nm. EI MS spectra were recorded with a Thermo Trace 1300-ISQ GC/MS instrument  and m/z values are given along with their relative intensities (in %) at an ionization voltage of 70 eV. High-resolution mass spectra were recorded with a Bruker Daltonics (ESI microTOF-QII) mass spectrometer. IR spectra were recorded with a Nicolet iS50 FTIR spectrometer under ATP mode. Analytical samples were dried at 40–100 °C under reduced pressure ( ≈ 10−2 mbar). Melting points were measured with a Büchi Melting point M-560 apparatus and are uncorrected. Elemental analyses were obtained with a Vario MicroCube CHNS analyser. The values are expressed in mass percentage. The NMR spectra of all previously unpublished molecules are given in Suppl. Figs. All NMR spectra were recorded on a Bruker Avance 500 spectrometer at 25 °C in CDCl34. Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac molecules were deposited by using a spray deposition technique onto a Au(111) substrate32 cleaned by repeated cycles of sputtering and annealing. Therefore, the clean Au(111) crystal is placed directly opposite to a pinhole in a rough vacuum chamber ( ≈ 1 × 10−2 mbar). About 1 μL of molecular solution (1 mg of molecules in 1 mL of dichloromethane) is being sucked in through the pinhole so that small droplets of the solution land on the sample32, see Suppl. Fig. −10 mbar).Low-temperature STM and STML measurements were performed using a homemade UHV STM with optical access operated at 4.4 Konto a Au11 substr34. Light emitted from the junction is guided into an optical fiber (core diameter 200 μm) via a parabolic mirror microfabricated by using 3D direct laser writing. The fiber goes into a spectrometer  equipped with a grating of 300 groves mm−1. The optical resolution of this setup is about 8 nm when the entrance slit is fully open (400 μm) and can be reduced to about 2 nm when the entrance slit is closed to 10 μm.Our STM is equipped with optical access to collect the light emitted from the tunneling junctionAll photon spectra presented in the manuscript are corrected for the collection efficiency of the detector (see Suppl. Fig. The plasmonic response of the mirror tip used in this work shows a single broad peak centered around 2 eV (see Suppl. Fig. Tol-Tpd-sNDI-Ac and Tol-Tpd-nNDI-Ac were pre-optimized using the r2SCAN functional49 in combination with the def2-TZVP basis set50. Excitation spectra were then obtained using the TMHF ab initio local hybrid functional51. Energy and density thresholds were set to 10−8 and 10−6 a.u. respectively. A grid of size “veryfine” was used52. All DFT calculations in vacuum were carried out using TURBOMOLE V7.753.The structures of Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Reporting Summary


Reducing local impacts can increase reef resistance to and recovery from bleaching2. However, resource managers lack clear advice on targeted actions that best support coral reefs under climate change3 and sector-based governance means most land- and sea-based management efforts remain siloed4. Here we combine surveys of reef change with a unique 20-year time series of land–sea human impacts that encompassed an unprecedented marine heatwave in Hawai‘i. Reefs with increased herbivorous fish populations and reduced land-based impacts, such as wastewater pollution and urban runoff, had positive coral cover trajectories predisturbance. These reefs also experienced a modest reduction in coral mortality following severe heat stress compared to reefs with reduced fish populations and enhanced land-based impacts. Scenario modelling indicated that simultaneously reducing land–sea human impacts results in a three- to sixfold greater probability of a reef having high reef-builder cover four years postdisturbance than if either occurred in isolation. International efforts to protect 30% of Earth’s land and ocean ecosystems by 2030 are underway5. Our results reveal that integrated land–sea management could help achieve coastal ocean conservation goals and provide coral reefs with the best opportunity to persist in our changing climate.Coral reef ecosystems are being fundamentally restructured by local human impacts and climate-driven marine heatwaves that trigger mass coral bleaching and mortality Surveys of reef change are combined with a unique 20-year time series of land–sea human impacts and the results show that integrated land–sea management could help achieve coastal ocean conservation goals and provide coral reefs with the best opportunity to persist in our changing climate. But with four times the population density living within 20 km of the ocean compared to the rest of the world7, direct human impacts on local scales are fundamentally restructuring these important marine communities8. Coastal areas are also affected by stronger and more frequent disturbances fuelled by human-induced climate change9. These human stressors are especially acute on tropical coral reefs where up to 90% of the local population live along the shoreline10. Land-based stressors, such as wastewater pollution, combine with sea-based stressors, such as overfishing, to disrupt natural ecological feedbacks on reefs11. Corals are further stressed by prolonged periods of anomalously warm ocean temperatures, known as marine heatwaves12, that can cause mass coral bleaching13 and mortality and fundamentally transform reef assemblages15.Coastal areas contain some of the most biologically diverse and productive marine ecosystems on Earth3. Its importance was established in the indigenous stewardship of island ecosystems, which used a decentralized and integrated resource management strategy that extended from the mountains to the sea17. By contrast, contemporary centralized governance means most terrestrial and ocean management efforts remain siloed18. As a result, whereas local resource managers have aspired to an integrated land–sea approach19, evidence of its efficacy above either approach in isolation remains wanting and difficult to test. Detecting conservation benefits in highly dynamic ecosystems is challenging20, but recent studies have identified salient connections between local conditions and coral reef resistance to and recovery potential following mass bleaching23. Managers therefore require unambiguous targets for the combination of land–sea human impacts they should mitigate to support coral reef persistence under climate change. Hampering these efforts are a lack of spatially resolved data on local drivers of coral reef ecosystems over time. Researchers are often forced to use proxies such as population density25 and reef accessibility26, or composite indices such as ‘water quality’11 that can be affected by anything from deforestation27 to aquaculture28. Such proxies do not identify the policy levers local resource managers can pull and are less likely to result in management actions or successful conservation outcomes.Reducing human impacts on local scales to maintain ecosystem integrity has been the guiding model of coral reef conservation for decades31  and changed by less than 3% in the subsequent years leading up to the 2015 marine heatwave  greater on reefs with positive trajectories compared to those with negative trajectories . This finding supports the notion that human population density is a poor indicator of human-driven land–sea impacts at local scales33. We observed minimal differences between positive and negative trajectory reefs relative to fishing gear restrictions, depth, sediment input, ocean temperatures, phytoplankton biomass and rainfall. Wave exposure was slightly higher (8.6 kW m−1) on reefs with positive trajectories, but the difference is minor because the entire study region is generally protected from large wave events34.Coral cover among reefs surveyed in 2003 was 36.9 ± 2.3%  lost more than 20% coral cover whereas the hardest-hit reef lost 49%  among reefs one year after the 2015 marine heatwave in Hawai‘i  during the marine heatwave showed reduced coral mortality , high levels of chlorophyll-a are an indicator of poor water quality that drives negative outcomes for corals40. Here, chlorophyll-a was uncorrelated to land-based human impacts  is more indicative of coral reef recovery potential following disturbance. Our surveys four years following the 2015 marine heatwave found that reef-builder cover ranged from 3.4 to 51.9%  in predicting whether a reef had relatively higher reef-builder cover four years postdisturbance . Furthermore, our scraper biomass estimates were derived from multiple observations across several time points following the marine heatwave, rather than a single snapshot estimate. Such strong site-based fidelity, combined with our recurring surveys, suggests that resident scrapers played a key role in promoting higher reef-builder cover rather than the association driven purely by an influx of individuals seeking more favourable habitat postdisturbance.We used an ordinal logistic regression framework to identify the local land–sea human impacts and environmental factors that best supported coral reef persistence in the years following the 2015 marine heatwave. Decreased wastewater pollution and increased scraper biomass were the most important and significant (18. We generated management scenarios of how varying scraper biomass (sea-based management) and wastewater pollution (land-based management) influenced the probability of being in a low, moderate (more than the 25th and less than the 75th percentile) or high reef-builder cover category. Our findings indicate that an integrated management approach can result in a positive synergistic outcome for coral reefs  and relatively high wastewater pollution  is most likely to have low reef-builder cover (83% probability)  but wastewater pollution remains high, there is a 70% probability of moderate reef-builder cover (scenario A). Conversely, where wastewater pollution is lower , but scraper biomass remains low, there is an 83% probability of moderate reef-builder cover (scenario B). However, if both land and sea management scenarios occur, there is an 80% probability of high reef-builder cover (scenario C). Combining land and sea management resulted in a three- to sixfold increase in the probability of high reef-builder cover four years following severe heat stress than if land or sea were managed in isolation.Sea-based management efforts are often disconnected from those occurring on landefs Fig. . For exa30. Similarly, land-based impacts consistently emerged as driving negative coral reef outcomes, but the combination of stressors changed depending on the observational time window in question. Highly resolved data on the local human impacts that drive reef ecosystem trajectories over time are unlikely to be available in most regions. However, our overarching finding that integrated land–sea management benefits coral reefs under ocean warming, is applicable to populated reefs globally.Here we show that simultaneously mitigating local land- and sea-based human impacts promotes coral reef persistence before, during and in the years following a historically unprecedented marine heatwave in Hawai‘i. Our unique spatially and temporally resolved data highlighted the specific impacts that best correlated with coral reef persistence in each of these temporal periods. For example, the biomass of all reef-fish groups was associated with positive reef trajectories over the 12 years leading up to the marine heatwave. By contrast, scraper biomass was the only fish group associated with positive outcomes for reefs four years following severe heat stress. This suggests that reef fish play essential functional roles at different points in time and that particular feeding and behaviours are probably critical for reef persistence following acute distrubance51. Distal human drivers also underpin climate change that is driving severe marine heatwaves that trigger mass coral bleaching at global scales. Increases in future ocean temperatures and the frequency and severity of coral bleaching events52 could simply overwhelm the positive effects of local management actions on coral reefs. However, there is substantial variation in the projected rates of ocean warming within and among countries under reduced emissions scenarios52. Actions that support coral reef persistence locally alongside global reductions in greenhouse gas emissions may buy reefs more time to adapt and persist into the future. Contemporary governance must therefore shift towards an integrated approach to align management strategies with reef ecosystem processes and the coincident multiscale human drivers that affect them1.The local human impacts we identify here represent the direct or proximate drivers of reef condition in our study. These in turn are dictated by an array of distal socioeconomic and cultural factors such as human migration and urbanization, finance, trade and tourism that indirectly affect how people interact with coral reefs5. The motivation behind the ‘30 by 30’ is to support ecological resilience, conserve biodiversity and preserve ecosystem services that underpin human well-being53. The 30 by 30 has broad participation and is being incorporated into conservation efforts by nations globally. However, our results reveal that sea-based management alone is insufficient to mitigate the full spectrum of local human effects on coastal ecosystems such as coral reefs. These efforts must therefore explicitly couple the respective 30% land–sea targets to realize coastal ocean conservation goals. But in most coastal geographies, 30% protection is impractical and unethical given the high proportion of people that live near and depend on these ecosystems54. Instead, mitigating land-based impacts such as wastewater pollution must occur together with fisheries governance for successful conservation outcomes, akin to long-standing indigenous stewardship practices of island ecosystems16. Only by adopting coupled land–sea policy measures, alongside global emissions reductions, will coral reef ecosystems and the human communities they support have the best opportunity for persistence in our changing climate.An ambitious effort is underway to protect 30% of Earth’s land and sea areas by 2030 as part of the recently adopted Kunming-Montreal Global Biodiversity Framework55 and large gradients in human population, local land–sea impacts and environmental factors that are comparable to reef ecosystems globally  is the southeastern most island of the Hawaiian Archipelago, located in the northern central Pacific . Between 30 and 50 random points were overlaid on each photograph and the benthic component under each point was identified to the lowest possible taxonomic level. Percentage cover of the major functional groups at each reef were used in this analysis, namely hard coral and crustose coralline algae. Surveys of reef-fish assemblages were performed along the same permanently marked 25 m transects concurrently with benthic surveys. However, reef-fish surveys were performed more frequently (one to six times per year from 2003 to 2019) than benthic surveys, depending on the reef location and monitoring programme performing the surveys. In all surveys, fishes were identified to species, sized and enumerated. To account for differences among programmes in how researchers surveyed reef fish, counts were calibrated using species and method specific adjustments56.Full details related to sampling design, site selection and survey frequency for benthic and reef-fish data collection across our study region are in the 56. Total fish biomass is an indicator of the overall state of the fish assemblage57 and is reduced in areas that have increased fishing pressure59. In Hawai‘i, non-commercial nearshore fisheries dominate, with people fishing for recreational, subsistence and cultural purposes61. However, the dominant harvesting modes and magnitude of fishing activities are largely unknown at spatial or temporal scales relevant to this study62. As such, we include total fish biomass in part to represent fishing effort on reefs but recognize its shortcomings in capturing reef- and species-specific differences in fishing pressure across our study region. We also include herbivores and subdivisions by feeding guilds that represent important indicators of resilience on coral reefs64. Browsers are defined as herbivores that feed on macroalgae and associated epiphytic material, and are important for reducing the cover of larger, more established macroalgae. Grazers are herbivores that feed largely on small algal turfs, helping to prevent their succession into larger macroalgae, and scrapers are herbivores that closely crop the substrate and open up new space to promote the settlement, growth and survival of crustose coralline algae and corals30.The biomass of fishes at a given reef was measured as total fish biomass, herbivore fish biomass and the biomass of browsers, grazers and scrapers56. The biomass of individual fishes was estimated using the allometric length–weight conversion: W = aTLb, where parameters a and b are species-specific constants, TL is total length (cm) and W is weight (g). Length–weight fitting parameters were obtained from a comprehensive assessment of Hawai‘i specific parameters56 and FishBase65. Fish species were excluded from fish biomass calculations according to life history characteristics that are not well captured with visual surveys, including cryptic benthic species, nocturnal species, pelagic schooling species and manta rays.We followed established methods for calculating fish biomass66). The dataset is available at 1 km resolution at 5-year intervals. Linear interpolation was used to fill in the missing years and produce annual time steps of human population within 15 km of each 100 m grid cell across our study region  and nitrogen input (kg ha−1 yr−1) from onsite sewage disposal systems  and injection wells (collectively OSDS) in coastal waters at 100 m resolution. Only OSDS located within a modelled one-year groundwater travel time of the coast were included in the analysis and nutrients from OSDS were assumed to flow to the nearest point on the shoreline. Wastewater effluent and nutrient input were estimated on the basis of ref. 67 and discharge rates and nutrient loading according to ref. 68. A Gaussian decay function was used to estimate dispersal offshore, approaching zero at 2 km  at 100 m resolution as the combination of total nitrogen from OSDS (−1 (refs. 70) and then by a leaching rate of 32%73 to estimate nitrogen that either runs off or reaches the groundwater. We also imposed a reduction in nitrogen that reached the ocean on the basis of distance inland and used subwatershed catchment data74 to estimate nutrient transport from golf courses to the coastline  within 10 km of the coastline at 100 m resolution for each year from 2000 to 2017  and peak rainfall  at 100 m resolution. Daily rainfall data were generated following refs. 76. Rainfall from each rain station was used to derive interpolated surfaces at annual time steps using Empirical Bayesian Kriging in ArcGIS. Subwatershed catchment data74 were clipped to 0–10 km from the coast and used to calculate rainfall per drainage area  reaching the coast80 at 100 m resolution. We then modulated the long-term annual average sediment over time by watershed on the basis of discharge calculated from peak rainfall data  full no-take, (2) no lay net, spear or aquarium, (3) no lay net or aquarium, (4) no lay net, (5) no aquarium and (6) open to all gear types  in summertime sea surface temperature (SST) was calculated over a 90-day window centred on the maximum value of a 7-day moving window average for each SST pixel  and irradiance (E m−2 d−1) from two sources. The long-term mean (2002–2013) in 8-day, 4 km data were obtained from ref. 80 and shown in Fig. 83  combines wave height and period and provides a more representative metric of wave exposure than wave height alone84. A series of nested grids  using WAVEWATCH III85 and Simulating Waves Nearshore86 were used to quantify wave transformation over the reef environment at 50 m, at hourly intervals across our study region from ref. 87 and updated for this study. Annual data were then generated for each 50 m grid cell by taking the mean of the top 97.5% in daily maximum wave power  was measured using diver depth gauges during the in-water reef surveys.We quantified the change in coral cover at 23 reefs from 2003 to 2014. A reef was considered to have a positive trajectory or negative trajectory if coral cover from the 2003 survey to the 2014 survey increased or decreased by greater than 3%, respectively Fig. . This cu88. Upper and lower bars in Fig. 89 based on a Euclidean distance similarity matrix, type III  sums-of-squares and unrestricted permutations of the normalized data. We visualized the results in Fig. 90 and calculated the cross-validation allocation success (a measure of group distinctness) from the leave-one-out procedure of the constrained analysis of principal coordinates analysis.The difference in local human impacts and environmental factors between positive and negative trajectory reefs were then calculated as the difference in the mean drop-one jackknife values for each impact or factor91  or 2015 (n = 40) and 2016 (n = 80); human population, wastewater pollution, nutrient loading, urban runoff, annual rainfall, peak rainfall and wave exposure were taken from the mean of all data from 2012 to 2016, sediment was measured from the mean of the top three events from 2006 to 2016; SST mean and SST variability were taken from the mean from 2000 to 2014; DHW was the maximum value for 2015; phytoplankton biomass and irradiance was the mean from June to November 2015, representing the time inclusive of the marine heatwave; fishing gear restrictions was the marine managed area designation before the marine heatwave  and depth came from in-water diver-assessed values.We then calculated the following predictors based on current literature and our hypotheses of the principal factors that drive changes in coral cover owing to severe heat stress (Extended Data Table 24 with the gamm4 (ref. 93) package for R (www.r-project.org) v.4.0.2. Before model fitting, we identified the presence of outliers in our predictor variables as any point that fell outside a threshold of ±2 standard deviations of the median. We then applied an additional step to retain any point above this threshold that was within 25% of the maximum predictor value below the threshold. This ensured that no data points were unnecessarily discarded from our formal model-fitting process because of applying an arbitrary threshold cut-off for data inclusion. The following predictors were square-root transformed to down-weight the influence of values at the extreme ends of their distributions: all fish biomass metrics, wastewater pollution, urban runoff, nutrient loading, phytoplankton biomass and peak rainfall. A fourth-root transformation was applied to sediment.We then tested for correlations between coral loss and our suite of predictor variables using a generalized additive mixed-effects modelling (GAMM) frameworkr > 0.7) predictors. To further strive for model parsimony, we a priori excluded human population density from the model-fitting process as it was a poor indicator of human-driven land-to-sea impacts on local scales  spread evenly throughout each covariate. All possible candidate models were computed (unique combinations of the predictor variables) but limiting the total number of predictors in any given candidate model to five to reduce overfitting. We used Akaike’s information criterion with a bias correction for small sample sizes95 (AICc) for model comparison and all models within ΔAICc ≤ 2 of the top model (ΔAICc = 0) are presented in Extended Data Table We incorporated a random spatial factor to account for the possible influence of a change in an underlying variable along the coastline not quantified in this study. This was done by breaking the coastline up into discrete 10 km sections running north to south. Section size was determined using hierarchical clustering based on pairwise Euclidean distances between reefs and identifying an inflection point in the intragroup variancen = 55) were parsed into three categories on the basis of the following percentiles: low, less than or equal to the 25th; moderate, more than 25th and less than 75th; and high, more than or equal to the 75th. We then performed ordinal logistic regression96 to determine the probability of a given reef having high, moderate or low cover of reef-building organisms on the basis of the prevailing local human impacts and environmental factors  or 2 (moderate). Reefs with low reef-builder cover contributed to the regression through calculation of the log odds. Each Cj is an MLE-computed model intercept, and each Bk is the MLE coefficient corresponding to the standardized independent variable zik, for k = 1 through n, where n is the variable number of predictors used in a given candidate model, hence the ellipsis (...). A fundamental component of this model is the assumption of proportional odds, or parallel regression, which indicates that Bk values are independent of the logit level j. The validity of this parallel regression assumption was ascertained using Brant’s Wald test97, as well as a likelihood ratio test (α = 0.05).Our goal was to assess the local land–sea human impacts and environmental factors that best explained variations in the cover of reef-building organisms four years following the marine heatwave. The cover of reef-building organisms for reefs surveyed in 2019  predictors. For the reasons outlined in our GAMM analysis and for continuity, we a priori excluded human population density and the biomass of browsers from the model-fitting process. This resulted in the following predictors included in the models (correlated predictors in parentheses were removed): total fish biomass, biomass of scrapers, biomass of grazers , wastewater pollution, nutrient input, sediment input, urban runoff (phytoplankton biomass), wave exposure, fishing gear restrictions and depth. The decision of which correlated predictors to retain followed the same logic as our GAMM analysis. The mean and variability in SST were excluded given the negligible range of values among reefs . All possible candidate models were computed while limiting the total number of predictors in any given candidate model to four . Models were computed using the multinomial logistic regression function mnrfit in MATLAB. We again used AICc for model comparison and all models within ΔAICc ≤ 2 of the top model (ΔAICc = 0) are presented in Extended Data Table R2 was computed for the highest ranked models and ranged from 0.21 to 0.22. Unlike traditional R2 values, McFadden’s pseudo-R2 of more than 0.2 represents an excellent fit98. Models within ΔAICc ≤ 2 of model 1 in Extended Data Table 100. Many of the parameter coefficients within these models were sensitive to the underlying variability in the data and their estimates did not differ significantly from zero (P < 0.05). The top model contained parameters with covariate estimates significantly different from zero, namely scraper biomass and wastewater pollution. Using model 1, we examined changes in the probability of a given reef having high , moderate (more than the 25th and less than the 75th percentile) or low  reef-builder cover . We then square-root transformed the following predictors to down-weight the influence of values at the extreme ends of their distributions: total fish biomass, wastewater pollution, sediment input and nutrient loading. Pearson’s correlation coefficients were calculated among all predictors  biomass of scrapers within Kealakekua Bay, a marine protected area where no fishing has been allowed since 1969 . As such, our value of 250 kg ha−1 represents an estimate of scraper biomass on a reef with strong fisheries protection but with land-based stressors present. In addition, we compared our upper (250 kg ha−1) and lower (30 kg ha−1) scraper biomass values to the distribution of scraper biomass among all reefs (n = 80) in 2019, the most recent time point in which all reefs were surveyed within the same year  and lower  values chosen for wastewater pollution represented the 95th and 36th percentiles of the 2019 distribution, respectively (Supplementary Fig. The resource management scenarios presented in Fig. Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06394-w.Supplementary InformationReporting SummaryPeer Review File