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DOE OSTI2020

Burrowing Owl Conservation Report: 2020

The Western Burrowing Owl (Athene cunicularia) is a small migratory species of owl found throughout the Western United States. Burrowing Owls are found throughout a variety of habitats including shrubsteppe; and during the breeding season the Hanford Site is situated at the center of the predicted distribution of Burrowing Owls in Washington State (Smith et al. 1997). The Western Burrowing Owl is declining over much of its range, including on the Hanford Site and throughout the Columbia Basin (HNF-59375, Conway 2006). These declines have been attributed to habitat loss and a subsequent loss in burrowing mammals that the owls depend on for nesting habitat (Conway et al. 2006, Johnson et al. 2010). The Washington State Department of Fish and Wildlife lists Western Burrowing Owls as a Candidate Species, meaning the species is a candidate for listing as Sensitive, Threatened, or Endangered in Washington (WDFW 2020). Without intervention, continued declines may result in the extirpation of Burrowing Owls from the Hanford Site and the Columbia Basin.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Summer Bat Monitoring Report for Calendar Year 2024

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Summer Bat Monitoring Report for Calendar Year 2023

Bats are mammals belonging to the order Chiroptera, which means “hand-wing.” They use a thin membrane of skin stretched between their fingers to fly. All bat species known in the state of Washington are insectivorous and each bat can consume 600-1,000 insects per hour while feeding. Bats typically give birth once per year to a single pup, making bat populations vulnerable to impacts and slow to recover. Several species of bats have been documented on the Hanford Site, with nine species identified during The Nature Conservancy surveys in 1997 and 1998, and an additional seven species listed as potentially present as documented in Biodiversity Inventory and Analysis of the Hanford Site. Final Report 1994-1999 (Soll et al., 1999). Ongoing U.S. Department of Energy (DOE), Richland Operations Office (RL) acoustic surveys, including HNF-53759, Summer Bat Monitoring Report for Calendar Year 2012, have also confirmed the presence of the same nine species documented by Soll et al. (1999). An additional species, the spotted bat (Euderma maculatum), was observed roosting on the exterior of a facility by Hanford Site personnel in 2021; the species was confirmed by Hanford Site biologists via photograph (Table 1-1).

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Towards Commercialization of Hydrophone Flow Shields to Improve Passive Acoustic Data Quality (CRADA 678) (Final Report)

This project performed testing, market research, and manufacturing refinement to ready patent-pending hydrophone flow shields developed at PNNL for commercialization. The flow shields reduce the impact of flow noise on underwater sound recordings in areas of strong flow, like tidal channels or rivers. Flow noise is a challenge in many applications where it is necessary to measure underwater sound, including measuring the sound produced by marine energy converters and monitoring marine mammals. This project addressed several key questions about flow shield performance that are high-priority for potential customers and refined existing manufacturing processes to be scalable for market-level production. Specifically, this project conducted both laboratory and field evaluations of the flow shields to comprehensively evaluate their performance. Overall, the results indicate that the flow shields reduce flow noise by over 20 dB at frequencies below 50 Hz at flow speeds greater than 0.5 m/s and are proven to survive deployments more than 6 months long.

42 ENGINEERING↗

DOE OSTI2022

U.S. Pacific Coast Workshop Report on Preconstruction Research Recommendations (U.S. Offshore Wind Synthesis of Environmental Effects Research (SEER) Project)

In May 2022, the U.S. Offshore Wind Synthesis of Environmental Effects Research (SEER) project team hosted a stakeholder workshop focused on preconstruction (baseline) research needs for potential floating offshore wind (OSW) energy development on the U.S. Pacific Coast, including California, Oregon, and Washington. Prior to the workshop, the SEER team developed a set of initial synthesized research recommendations that were identified based on a review of relevant, publicly available resources and with advisory group input. The workshop covered three marine life breakout groups on subsequent days to discuss research recommendations related to 1) marine mammals and sea turtles, 2) fish and invertebrates, and 3) birds and bats. As part of the workshop, over a hundred participants from the public and private sectors provided feedback on various aspects of the initial research recommendations, including associated data and knowledge gaps, benefits/limitations of available methods and technologies, and technological advancements or infrastructure needed to address the recommendation. Approximately 1,000 total comments were received on the workshop MURAL boards and were synthesized in this report. Based on workshop feedback, SEER developed a final database of over 500 specific research recommendations based on more than 40 resources. In Fall 2022, the full database and a tool with updated synthesized research recommendations were disseminated on Tethys (https://tethys.pnnl.gov) to assist with informing future funding opportunities and research programming. There is a continued need to improve awareness of the potential environmental effects, monitoring technologies, and management strategies for floating OSW energy development on the U.S. Pacific Coast. Coordination of these activities will require the sustained involvement of multiple stakeholders from across sectors. Beyond the baseline considerations discussed in this workshop, future state-of-the-science activities should be planned to consider research needs across wind energy life cycle phases for all relevant wildlife taxa and associated habitat and ecosystem processes.

17 WIND ENERGY↗

DOE OSTI2024

N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N α -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5 , N 5 -di-methyl N α -acetyl-L-ornithine (k cat /K m = 7.32 ± 0.94 × 10 4 M -1 s -1 ). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC 50 = 58.7 μM; K i = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC 50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K i = 35.9 ± 5.1 μM). The active site structure of di-Zn Ec ArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Fencing affects movement patterns of two large carnivores in Southern Africa

The erection of peripheral fences around protected areas has been shown to adversely affect the movement behavior, genetic connectivity and long-term viability of animal populations. However, fencing is also used extensively to reduce human encroachment, limit poaching activities, and mitigate human–wildlife conflicts. Limited information on the movement behavior of carnivores is available along the periphery of protected areas, particularly in semi-arid environments. To quantify the potential effects of the 824 km perimeter fence surrounding the Etosha National Park (Etosha, Namibia) on the movement and space use of carnivores, we collected GPS movement data from 36 lions (Panthera leo) and seven spotted hyenas (Crocuta crocuta) over a 14-year period. For these two apex carnivores, we measured the impact of the Etosha fence on their movements (step lengths and path straightness), calculated the likelihood of fence crossings, and evaluated the spatial–temporal characteristics of these crossings. Our results indicated that lions and hyenas moved faster (longer step lengths) when closer to the fence. Lions showed no variation in path straightness with respect to the fence but hyenas had straighter movement paths when closer to the fence. When moving within the vicinity of the fence, lions had a 9% likelihood of crossing the fence, while hyenas were much more likely (18%) to do so, which suggests that the Etosha fence was more permeable for hyenas than for lions. Fence crossings predominantly occurred at night for both species. Lions were more likely to cross the fence during the cold dry season (May–August) whereas hyenas crossed more often during the warm wet season (December–April). Female and adult lions had a higher probability of crossing the fence than males and subadults. These findings could be useful in the development of appropriate strategies to mitigate human–carnivore conflicts and thus promote the conservation of carnivores in multiple-use areas along the periphery of the park. Further research is needed to better elucidate the impact of perimeter fences on a broader suite of carnivores and other large mammals, including species-specific variation in the response to fences, as well as the economic benefits of fencing provided to local communities.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

The Structure and Immune Regulatory Implications of the Ubiquitin-Like Tandem Domain Within an Avian 2’-5’ Oligoadenylate Synthetase-Like Protein

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host’s ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2’-5’ oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Comparison of gene expression in the skin tissue of gray, humpback, and fin whales

Analyses of gene expression in the skin of several species of whales identified genes that are differentially expressed in association with environmental factors, suggesting that skin transcriptomics may provide a valuable tool for assessing physiological responses in marine mammals. Previous work exploring differing levels of gene expression has focused on odontocetes with comparatively limited investigation of skin gene expression has been explored in mysticetes. Here, we describe the identity of genes expressed in skin tissue of three species of baleen whales to establish a baseline of gene expression and compare gene identity and expression patterns across species. We also evaluate sex-specific differences in skin gene expression through a comparison of expression levels between males and females in gray and humpback whales. A total of 16 skin tissue samples were collected from free-ranging gray, humpback and fin whales off the central Oregon coast in the eastern North Pacific. Comparison of the expressed genes in the humpback and gray whale skin tissue to the blue whale reference database identified enriched gene ontology terms in the skin tissue of each species, suggesting genes over-represented in the whale skin related to cell epithelial development, regulation of gene expression and cell maintenance . Comparison of gene expression between male and female samples revealed sex-specific differences in gray and humpback whales. A differential gene expression analysis identified several x-linked genes that have been previously identified and show gene expression differences in male and female cetaceans, such as ZFX, DDX3X and USP9X. Establishing baseline skin gene expression profiles for these three baleen whale species sampled off the Oregon coast provides a foundation for linking transcriptome variation with physiological condition and environment.

Sremba, Angela↗

DOE OSTI2023

Extracellular electron uptake from a cathode by the lactic acid bacterium Lactiplantibacillus plantarum

A subset of microorganisms that perform respiration can endogenously utilize insoluble electron donors, such as Fe(II) or a cathode, in a process called extracellular electron transfer (EET). However, it is unknown whether similar endogenous EET can be performed by primarily fermentative species like lactic acid bacteria. We report for the first time electron uptake from a cathode by Lactiplantibacillus plantarum, a primarily fermentative bacteria found in the gut of mammals and in fermented foods. L. plantarum consumed electrons from a cathode and coupled this oxidation to the reduction of both an endogenous organic (pyruvate) and an exogenous inorganic electron acceptor (nitrate). This electron uptake from a cathode reroutes glucose fermentation toward lactate degradation and provides cells with a higher viability upon sugar exhaustion. Moreover, the associated genes and cofactors indicate that this activity is mechanistically different from that one employed by lactic acid bacteria to reduce an anode and to perform respiration. Our results expand our knowledge of the diversity of electroactive species and of the metabolic and bioenergetic strategies used by lactic acid bacteria.

biocathode↗

DOE OSTI2024

Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens

Introduction Control ofCampylobacterfrom farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs ofCampylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allowsCampylobacterto colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO 4 ). Methods Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigateCampylobacterin infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO 4 (1 mM) inC. jejuni81–176, a human isolate which is widely studied. Results Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO 4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1–T8) were selected for further studies. These selected eight Tat inhibitors cleared all testedCampylobacterstrains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO 4 in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellularC. jejunicells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO 4 . Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log;p < 0.01) with minimal disruption of microbiota.In silicoanalyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein. Discussion Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to controlC. jejunispread on farms.

Microbiology↗

DOE OSTI2024

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2 . Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

Dai, Jiage↗

DOE OSTI2021

Consumption Patterns of a Generalist Omnivore: Eastern Box Turtle Diets in the Long Island Pine Barrens

Eastern Box Turtles (Terrapene carolina) are diet generalists and as such are predicted to have diverse diets in which familiar, low-quality foods are eaten consistently at low levels, and high-quality foods are rare but eaten whenever available. Previous work showed that they feed opportunistically on seasonally available plants (shoots, leaves, flowers, and fruit), invertebrates, mushrooms, and occasionally carrion. We used fecal samples to test optimal foraging predictions relevant to diet generalists and also whether the Eastern Box Turtle diets varied seasonally in a northeastern U.S. pine-oak habitat. We found that in-depth prey species consumption patterns of six different individuals were similar to those of the sampled population overall. Leaf and stem material was consumed by 100% of the turtles in all months despite being lower-quality than other prey available. Invertebrates were consumed by at least 80% of turtles in every study period; Coleopterans were found more commonly than other invertebrates. Snails were not eaten by more than 20% of the turtles in any study period, and mushroom consumption varied from 31–75% of samples in different study periods. Monthly diet overlap was measured using both Pianka’s Index of Overlap (PIO) and the Morisita–Horn Index (MH). The PIO method indicated that the prey consumption patterns were broadly similar from June–October, while the M–H method showed that only the July vs. August comparison was highly similar. The turtle diets changed only slightly between seasons, and they conform to predictions of diet generalist models usually applied to mammals.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Marine Energy Environmental Permitting and Compliance Costs

Costs to permit Marine Energy projects are poorly understood. In this paper we examine environmental compliance and permitting costs for 19 projects in the U.S., covering the last 2 decades. Guided discussions were conducted with developers over a 3-year period to obtain historical and ongoing project cost data relative to environmental studies (e.g., baseline or pre-project site characterization as well as post-installation effects monitoring), stakeholder outreach, and mitigation, as well as qualitative experience of the permitting process. Data are organized in categories of technology type, permitted capacity, pre- and post-installation, geographic location, and funding types. We also compare our findings with earlier logic models created for the Department of Energy (i.e., Reference Models). Environmental studies most commonly performed were for Fish and Fisheries, Noise, Marine Habitat/Benthic Studies and Marine Mammals. Studies for tidal projects were more expensive than those performed for wave projects and the range of reported project costs tended to be wider than ranges predicted by logic models. For eight projects reporting full project costs, from project start to FERC or USACE permit, the average amount for environmental permitting compliance was 14.6%.

16 TIDAL AND WAVE POWER↗

DOE OSTI2022

Epigenetic Regulation by Polycomb Complexes from Drosophila to Human and Its Relation to Communicable Disease Pathogenesis

Although all cells in the human body are made of the same DNA, these cells undergo differentiation and behave differently during development, through integration of external and internal stimuli via ‘specific mechanisms.’ Epigenetics is one such mechanism that comprises DNA/RNA, histone modifications, and non-coding RNAs that regulate transcription without changing the genetic code. The discovery of the first Polycomb mutant phenotype in Drosophila started the study of epigenetics more than 80 years ago. Since then, a considerable number of Polycomb Group (PcG) genes in Drosophila have been discovered to be preserved in mammals, including humans. PcG proteins exert their influence through gene repression by acting in complexes, modifying histones, and compacting the chromatin within the nucleus. In this article, we discuss how our knowledge of the PcG repression mechanism in Drosophila translates to human communicable disease research.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Exploring the Interplay between Polyphenols and Lysyl Oxidase Enzymes for Maintaining Extracellular Matrix Homeostasis

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

The Gut Microbiome and Alcoholic Liver Disease: Ethanol Consumption Drives Consistent and Reproducible Alteration in Gut Microbiota in Mice

Phenotypic health effects, both positive and negative, have been well studied in association with the consumption of alcohol in humans as well as several other mammals including mice. Many studies have also associated these same health effects and phenotypes to specific members of gut microbiome communities. Here we utilized a chronic plus binge ethanol feed model (Gao-binge model) to explore microbiome community changes across three independent experiments performed in mice. We found significant and reproducible differences in microbiome community assemblies between ethanol-treated mice and control mice on the same diet absent of ethanol. We also identified significant differences in gut microbiota occurring temporally with ethanol treatment. Peak shift in communities was observed 4 days after the start of daily alcohol consumption. We quantitatively identified many of the bacterial genera indicative of these ethanol-induced shifts including 20 significant genera when comparing ethanol treatments with controls and 14 significant genera based on temporal investigation. Including overlap of treatment with temporal shifts, we identified 25 specific genera of interest in ethanol treatment microbiome shifts. Shifts coincide with observed presentation of fatty deposits in the liver tissue, i.e., Alcoholic Liver Disease-associated phenotype. The evidence presented herein, derived from three independent experiments, points to the existence of a common, reproducible, and characterizable “mouse ethanol gut microbiome”.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Toll-like Receptor Ligands Enhance Vaccine Efficacy against a Virulent Newcastle Disease Virus Challenge in Chickens

To enhance the efficacy of the current Newcastle disease vaccine, we have selected potential adjuvants that target well-characterized pattern recognition receptors: the toll-like receptors (TLRs). Imiquimod is a small-molecule activator of TLR7, which is a sensor of dsDNA. ODN-1826 is a mimetic of CpG DNA and ligates TLR21 (a chicken homologue of TLR9 in mammals). The activation of TLRs leads to antiviral responses, including the induction of type I interferons (IFNs). In this study, birds were vaccinated intranasally with a live LaSota strain with or without imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, the birds were challenged with a virulent Newcastle disease virus (chicken/CA/212676/2002). Both adjuvants (imiquimod or ODN-1826) induced higher and more uniform antibody titers among vaccinated birds compared with the live vaccine-alone group. In addition, adjuvanted vaccines demonstrated greater protective efficacy in terms of the reduction in virus-shedding titer and the number of birds shedding the challenge virus at 2 and 4 days post-challenge. A differential expression of antiviral and immune-related genes was observed among groups from tissues (Harderian gland, trachea, cecal tonsil, and spleen) collected 1 and 3 days after treatment. These results demonstrate the potential of TLR-targeted adjuvants as mucosal vaccine enhancers and warrant a further characterization of immune correlates and optimization for efficacy.

59 BASIC BIOLOGICAL SCIENCES↗