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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Abstract Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Complex decay dynamics of HIV virions, intact and defective proviruses, and 2LTR circles following initiation of antiretroviral therapy

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t 1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4 + T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t 1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ~3 mo, the decay slope changes, and CD4 + T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

2LTR circles↗
DOE OSTI2026

Genetic variations and their interaction with thirdhand smoke exposure on anxiety and memory in Collaborative Cross mice

Thirdhand smoke (THS) is linked to adverse health effects, but the effect of genetic variations on behavioral outcomes is poorly understood. To investigate this, we assessed anxiety- and memory-related behaviors in 820 mice from 21 strains of the genetically diverse Collaborative Cross (CC) mouse that were exposed to THS from 4 through 10 weeks of age. Anxiety was evaluated with a light/dark box assay with a previously established risk score system. Females were generally more sensitive: THS reduced anxiety risk in strains CC013, CC019, and CC051, but increased risk in CC036 and CC061, while males showed no significant effects. Memory was tested using passive avoidance: impairments were observed in both sexes in CC016 and CC019, with sex-dependent effects in CC002 and CC051. A genome-wide association study identified 2,347 SNPs associated with anxiety and 1,568 SNPs with memory, with 32 and 85 SNPs, respectively, interacting with THS exposure. Enrichment analyses revealed distinct biological processes underlying susceptibility, including axonogenesis, synapse organization, cognition, and learning and memory. KEGG pathway analysis identified distinct genetic pathways, including GTPase binding and GTPase regulatory activity, that act as critical molecular switches in the brain that regulate synaptic plasticity, dendritic spine structure, and neuronal signaling, directly influencing anxiety-like behaviors and memory formation. These findings show that THS exposure affects neurobehavioral outcomes in a sex- and genotype-dependent manner, highlighting critical gene-environment interactions and providing a foundation for mechanistic insights into THS neurotoxicity

Anxiety↗
DOE OSTI2022

Radius measurement via super-resolution microscopy enables the development of a variable radii proximity labeling platform

The elucidation of protein interaction networks is critical to understanding fundamental biology as well as developing new therapeutics. Proximity labeling platforms (PLPs) are state-of-the-art technologies that enable the discovery and delineation of biomolecular networks through the identification of protein-protein interactions. These platforms work via catalytic generation of reactive probes at a biological region of interest; these probes then diffuse through solution and covalently “tag” proximal biomolecules. The physical distance that the probes diffuse determines the effective labeling radius of the PLP and is a critical parameter that influences the scale and resolution of interactome mapping. As such, by expanding the degrees of labeling resolution offered by PLPs, it is possible to better capture the various size scales of interactomes. At present, however, there is little quantitative understanding of the labeling radii of different PLPs. Here, we report the development of a superresolution microscopy-based assay for the direct quantification of PLP labeling radii. Using this assay, we provide direct extracellular measurements of the labeling radii of state-of-the-art antibody-targeted PLPs, including the peroxidase-based phenoxy radical platform (269 ± 41 nm) and the high-resolution iridium-catalyzed µMap technology (54 ± 12 nm). Last, we apply these insights to the development of a molecular diffusion-based approach to tuning PLP resolution and introduce a new aryl-azide-based µMap platform with an intermediate labeling radius (80 ± 28 nm).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates

Animals produce bile to maximize absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also act as signaling molecules that bind host receptors and modulate physiological processes. Upon excretion of bile salts into the gut, the gut microbiome can create metabolites that have modified signaling capabilities. The category and magnitude of bile salt metabolism can affect the host either positively or negatively. A key modification is bile salt hydrolysis, which is a prerequisite step for all additional microbial modification. Here, we have synthesized five different fluorogenic bile salts that report on hydrolysis activity using a simple, continuous assay. We profile activity in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced bile salts, but some microbially modified bile salts also display significant turnover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

Abstract Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc‐dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl‐putrescine or ‐spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin‐labelled acetyl‐spermidine derivative to measure its PDAC activity, which is suitable for high‐throughput screening. Using this assay, we identified potent inhibitors of HDAC10‐mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10‐inhibitor structure using X‐ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Scripts for the manuscript "A fluorescence-based transient expression assay for the analysis of upstream open reading frames in plant" by Haas et al. Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome- wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild- type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost- efficient alternative to investigate transcript leaders by co- expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO2 assimilation during sun–shade transitions.

Gene Editing↗
DOE OSTI2025

Regulatory helix plays a key role in genetic ON-OFF switching for the 2’-deoxyguanosine sensing mRNA element

Transcriptional riboswitches, noncoding mRNA elements that operate in cis to regulate gene expression, have a promising potential in medicine, synthetic biology and directed evolution. They bind to cellular metabolites or metal ions with high specificity, leading to conformational rearrangements that facilitate the activation or premature termination of transcription for downstream genes. This elegant mechanism for feedback regulation of metabolic pathways has been identified in prokaryotes and a few in eukaryotes. Our chemical probing of the 2’-deoxyguanosine (2’-dG)-sensing riboswitch demonstrates that the overall conformational state of the full-length riboswitch (dGsw-fl) is unresponsive to the 2’-dG. Although binding proceeds as expected, dGsw-fl exclusively populates an OFF state of transcriptional inhibition. We chemically probed the structure of a known dGsw transcriptional intermediate (dGsw-int) to evaluate the possibility of a cotranscriptional regulatory role. Interestingly, apo dGsw-int adopts an alternative conformation in which a stable anti-terminator helix is formed, leading to an ON state where transcription can proceed. In the presence of 2’-dG, this anti-terminator helix is destabilized to produce a conformation reminiscent of the full-length, OFF-state dGsw. Using a fluorescence quenching assay, we demonstrate that binding 2’-dG to early transcriptional intermediates can inhibit the formation of the anti-terminator helix, locking dGsw in an OFF state. These data suggest that metabolite sensing occurs during a brief window of time between the synthesis of two transcriptional intermediates. Our studies indicate that dGsw does not function as a binary ON−OFF switch, but instead fine-tunes the transcription of downstream genes during RNA synthesis using key intermediates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Human sperm TMEM95 binds eggs and facilitates membrane fusion

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Magnetotactic Bacteria Accumulate a Large Pool of Iron Distinct from Their Magnetite Crystals

ABSTRACT Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that form intracellular nanoparticles of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) in a genetically controlled manner. Magnetite and greigite synthesis requires MTB to transport a large amount of iron from the environment. Most intracellular iron was proposed to be contained within the crystals. However, recent mass spectrometry studies suggest that MTB may contain a large amount of iron that is not precipitated in crystals. Here, we attempted to resolve these discrepancies by performing chemical and magnetic assays to quantify the different iron pools in the magnetite-forming strain Magnetospirillum magneticum AMB-1, as well as in mutant strains showing defects in crystal precipitation, cultivated at various iron concentrations. All results show that magnetite represents at most 30% of the total intracellular iron under our experimental conditions and even less in the mutant strains. We further examined the iron speciation and subcellular localization in AMB-1 using the fluorescent indicator FIP-1, which was designed for the detection of labile Fe(II). Staining with this probe suggests that unmineralized reduced iron is found in the cytoplasm and associated with magnetosomes. Our results demonstrate that, under our experimental conditions, AMB-1 is able to accumulate a large pool of iron distinct from magnetite. Finally, we discuss the biochemical and geochemical implications of these results. IMPORTANCE Magnetotactic bacteria (MTB) produce iron-based intracellular magnetic crystals. They represent a model system for studying iron homeostasis and biomineralization in microorganisms. MTB sequester a large amount of iron in their crystals and have thus been proposed to significantly impact the iron biogeochemical cycle. Several studies proposed that MTB could also accumulate iron in a reservoir distinct from their crystals. Here, we present a chemical and magnetic methodology for quantifying the iron pools in the magnetotactic strain AMB-1. Results showed that most iron is not contained in crystals. We then adapted protocols for the fluorescent Fe(II) detection in bacteria and showed that iron could be detected outside crystals using fluorescence assays. This work suggests a more complex picture for iron homeostasis in MTB than previously thought. Because iron speciation controls its fate in the environment, our results also provide important insights into the geochemical impact of MTB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex

MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Rewiring Aromatic Compound Consumption: Chromosomal Amplification and Evolution of a Foreign Pathway in Acinetobacter baylyi ADP1

Rational engineering strategies that seek to harness the remarkable diversity of microbial metabolism can be limited by incomplete biological knowledge. As described here, a novel approach to address this challenge involved replacing a native pathway for degrading lignin-derived aromatic compounds via ortho cleavage of protocatechuate in Acinetobacter baylyi ADP1 with a foreign meta-cleavage pathway that uses different enzymes, metabolites, and redox carriers. This alteration may improve lignin valorization and coordinate catabolism with bioproduction strategies. When a 14-kbp region of foreign DNA was inserted in the chromosome, the heterologous genes failed to confer growth on target substrates. Regional gene dosage was increased using a synthetic DNA fragment to promote recombination, and higher copy number enabled growth. During adaptive laboratory evolution, compensatory mutations arose that permit growth with one copy of the foreign genes. This complex metabolic remodeling was accomplished without assumptions about the impediments that initially prevented growth. To understand the changes that emerged, a novel transformation assay identified a combination of mutations sufficient for the new phenotype. Three unexpected changes were revealed: loss of one foreign enzyme, loss of one native enzyme, and loss of a two-component transcriptional regulatory system. This study establishes that large multicopy tandem arrays of poorly adapted pathway genes can confer new functions and improve understanding of metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A Bis(imidazole)-based cysteine labeling tool for metalloprotein assembly

Precise metal-protein coordination by design remains a considerable challenge. Polydentate, high-metal-affinity protein modifications, both chemical and recombinant, can enable metal localization. However, these constructs are often bulky, conformationally and stereochemically ill-defined, or coordinately saturated. Here, we expand the biomolecular metal-coordination toolbox with the irreversible attachment to cysteine of bis(1-methylimidazol-2-yl)ethene (“BMIE”), which generates a compact imidazole-based metal-coordinating ligand. Conjugate additions of small-molecule thiols (thiocresol and N-Boc-Cys) with BMIE confirm general thiol reactivity. The BMIE adducts are shown to complex the divalent metal ions Cu ++ and Zn ++ in bidentate (N 2 ) and tridentate (N 2 S*) coordination geometries. Cysteine-targeted BMIE modification (>90% yield at pH 8.0) of a model protein, the S203C variant of carboxypeptidase G2 (CPG2), measured with ESI-MS, confirms its utility as a site-selective bioconjugation method. ICP-MS analysis confirms mono-metallation of the BMIE-modified CPG2 protein with Zn ++ , Cu ++ , and Co ++ . EPR characterization of the BMIE-modified CPG2 protein reveals the structural details of the site selective 1:1 BMIE-Cu ++ coordination and symmetric tetragonal geometry under physiological conditions and in the presence of various competing and exchangeable ligands (H 2 O/HO – , tris, and phenanthroline). An X-ray protein crystal structure of BMIE-modified CPG2-S203C demonstrates that the BMIE modification is minimally disruptive to the overall protein structure, including the carboxypeptidase active sites, although Zn ++ metalation could not be conclusively discerned at the resolution obtained. The carboxypeptidase catalytic activity of BMIE-modified CPG2-S203C was also assayed and found to be minimally affected. Finally, these features, combined with ease of attachment, define the new BMIE-based ligation as a versatile metalloprotein design tool, and enable future catalytic and structural applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Physical and behavioral adaptations to prevent overheating of the living wings of butterflies

The wings of Lepidoptera contain a matrix of living cells whose function requires appropriate temperatures. However, given their small thermal capacity, wings can overheat rapidly in the sun. Here we analyze butterfly wings across a wide range of simulated environmental conditions, and find that regions containing living cells are maintained at cooler temperatures. Diverse scale nanostructures and non-uniform cuticle thicknesses create a heterogeneous distribution of radiative cooling that selectively reduces the temperature of structures such as wing veins and androconial organs. These tissues are supplied by circulatory, neural and tracheal systems throughout the adult lifetime, indicating that the insect wing is a dynamic, living structure. Behavioral assays show that butterflies use wings to sense visible and infrared radiation, responding with specialized behaviors to prevent overheating of their wings. Our work highlights the physiological importance of wing temperature and how it is exquisitely regulated by structural and behavioral adaptations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗