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Biology subjects

Kaplan, D. L.

Publications and source records attributed to Kaplan, D. L..

At least 19 recordsLinked to original sources

Molecular Engineering of Silk via Fatty Acid Esterification for Water-Stable, Anti-fouling, Biocompatible, Implantable Materials

Biopolymers offer attractive properties for implantable materials, but their limited aqueous and enzymatic stability constrains long-term performance in biological environments. Silk fibroin is mechanically tunable and thermoplastically processable, yet remains susceptible to swelling in aqueous environments, which can impact mechanical properties. Here, we establish fatty acid esterification as a strategy to transform silk into a hydrophobic, water-stable, yet still bioresorbable material (FAsilk). Fatty acid functionalization significantly increased resistance to aqueous swelling and enzymatic degradation while preserving structural and mechanical integrity and thermoplastic processability. The resulting FAsilk biomaterials also reduced microbial contamination, blood clot formation, and macrophage adhesion relative to native silk, due to the change in hydrophobicity, while maintaining cytocompatibility and hemocompatibility. Subcutaneous implantation in mice demonstrated favorable biological responses, including minimal impact on body weight and no significant pro-inflammatory responses, indicating no adverse effects on animal health. Thus, fatty acid functionalization enables silk to combine hydrophobicity and aqueous stability with processability and biodegradability, providing a mechanically robust materials platform for bioresorbable implants, including medical devices, tissue-engineered grafts and drug delivery systems.

bioengineering↗

Impact of Adipogenic Differentiation on the Nutrition and Sensory Profile of Cultivated Pork Fat Tissue

Adipose tissue is a principal source of texture, flavor, and aroma in meat products. Higher fat content corresponds to higher juiciness, tenderness and overall palatability of meat products, making it a key component of cultivated meat. Our previous study investigated the aromatic characteristics of cultivated pork fat tissue, finding comparable consumer panel hedonic scores between conventional livestock derived and cultivated pork fats. Further research was pursued to characterize if adipogenic differentiation of the cells is required to enhance the flavor of cultivated fat. While differentiation of cultivated cells results in tissues that more closely resemble the morphology of livestock-derived tissue, the process substantially increases production timelines and material costs associated with cultivated meat production. As a result, we observed changes in organoleptic and nutritional properties of cultivated pork fat cells associated with differentiation of porcine dedifferentiated fat (PDFAT) cells. We assessed the effect of varying degrees of adipogenesis with soybean oil supplementation on fat content, composition, and volatile compound formation. Notably, we observed the concentrations of 2-pentylfuran, 2-heptanone, hexanal and (E)-2-octenal increased compared to the undifferentiated preadipocytes. These compounds, along with other ketones, aldehydes, alcohol and lactone molecules, contribute to fruity, herbal, and fatty aromas. Additionally, these results were achieved without requiring the maximum differentiation time frame of 12 days in culture. These findings emphasize the importance of adipocyte differentiation in cultivated fat production and highlight its potential as a strategy for optimizing both flavor and nutritional attributes of cultivated meat.

bioengineering↗

Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

Fibrosis is a progressive and often fatal pathological process characterized by excessive extracellular matrix deposition, tissue stiffening, and irreversible organ dysfunction. Effective antifibrotic therapies remain limited by the lack of in vitro models that recapitulate the full spectrum of fibrotic disease progression. Here, we leverage tyramine-modified silk fibroin (SF-TA) hydrogels to investigate normal human lung fibroblasts (NHLF) responses to progressively stiffening environments relevant to pulmonary fibrosis. Two hydrogel formulations with distinct stiffening profiles over 14 days were prepared: a gradual-stiffening 0% SF-TA formulation reaching [~]20 kPa, and a rapidly stiffening 50% SF-TA formulation reaching [~]60 kPa. NHLFs were cultured on both formulations, with and without TGF{beta} (5 ng/mL), for 14 days and assessed for viability, metabolic activity, cytokine and collagen secretion, cytoskeletal organization, and mechanotransductive gene expression. The 0% SF-TA hydrogels drove sustained fibroblast proliferation and elevated secretion of IL-6, IL-8, and MCP-1, consistent with early inflammatory fibrosis. The 50% SF-TA hydrogels induced a metabolic plateau without senescence, suppressed inflammatory cytokine secretion, and, in the presence of TGF{beta}, led to significant upregulation of ACTA2 and CTGF, alongside -SMA stress fiber incorporation, consistent with established myofibroblast persistence. Both conditions produced comparable secreted collagen output by day 14. Together, these findings establish dynamically stiffening SF-TA hydrogels as a tunable platform for investigating stage-dependent fibroblast activation and mechanobiological progression in fibrosis.

bioengineering↗

Precision Fermentation of Recombinant Myofibrillar Proteins for Future Foods

Myofibrillar proteins, namely actin and myosin, are responsible for many of the textural attributes of animal-based meat. Precision fermentation (recombinant production of food ingredients) represents an underexplored approach to producing these proteins without the unsustainable practice of animal agriculture. We show that through the solubility-enhancing SUMO peptide tag and precipitation-based purification, we can produce actin via recombinant DNA methods at titers of 326 mg/L E. coli culture. We also show expression and precipitation of a recombinant fragment of the myosin tail, leading to 572 mg/L culture. For both proteins, yields are improved compared to prior studies, without the need for low-yielding laborious purification columns, with final purities of 69-73%. These recombinant actin and myosin proteins showed macro- and microscopic fibrous features similar to meat. When combined with plant-based proteins, chewiness, hardness, and Youngs modulus were improved towards that of animal-based meat. Preliminary cost analyses suggest a less expensive process for producing myofibrillar proteins compared to established methods. Our results reveal a novel scalable approach to making meat-like foods and ingredients through precision fermentation.

bioengineering↗

Engineered Vibrio natriegens lysate can replace multiple components of cell culture media

Reducing the cost and environmental impact of cell culture media is an important goal for cultivated meat, the process of generating meat in vitro using proliferating animal cells. While prior approaches have demonstrated the use of microbial lysates to replace expensive animal-based fetal bovine serum (FBS) in media, these formulations still rely on large quantities of growth factors such as fibroblast-like growth factor 2 (FGF2). Here, we demonstrate the use of FGF2-expressing Vibrio natriegens to create whole-cell lysates that replace both FBS and FGF2 in cell culture media for cultivated meat applications. This medium, named "VN40FGF", supports rapid proliferation of immortalized bovine muscle satellite cells (iBSCs) in the absence of supplemented FGF2. Cells grown in VN40FGF maintain phenotype and differentiation capacity. We also demonstrate that engineered V. natriegens can grow in spent cell culture media, further improving sustainability and economics, and reducing potential eutrophication concerns associated with waste disposal. Our approach combines multiple strategies for reducing the total number of media inputs, demonstrating opportunities for more economical and sustainable cell culture, especially for cultivated meats.

bioengineering↗

Fast structural search for classification of gut bacterial mucin O-glycan degrading enzymes

The Enzyme Commission (EC) numbering scheme provides a hierarchical way to classify enzymes according to their catalytic functions. While recent protein language model (PLM) based approaches like CLEAN and ProteInter have improved sequence-based EC number prediction, they struggle with fine-grained classification at the deepest hierarchical level. Structure-based approaches for grouping similar proteins using alignment tools excel at finding proteins that share overall global structure, but suffer from high false positive rates when classifying proteins that are globally structurally similar but functional differentiation depends on a localized region. This problem is particularly relevant to EC number prediction, as enzymatic function depends on its catalytic domain, which is a relatively small, specific region of the protein. We introduce Deep Enzyme Function Transfer (DEFT) that harmonizes sequence- and structure-based approaches through the key insight that PLM based annotations of the first two EC number hierarchy levels vastly reduce false positives that are likely to show in purely structure-based EC number prediction. Given an enzyme of interest, DEFT first uses a PLM based method to assign the first two levels of the enzymes EC number, and then uses a structure-based method to predict the remaining two levels of the EC number. Using benchmarking datasets, we demonstrate that DEFT achieves superior accuracy compared with current state-of-the-art tools for EC number prediction. Furthermore we show that DEFTs computational efficiency enables high-throughput, genome-wide annotations of total enzyme repertoires in organisms. We illustrate this capability by experimentally validating DEFT predicted glycoside hydrolase (GH) profiles of intestinal mucus associated bacteria. Author summaryEnzymes are ubiquitous proteins that catalyze chemical reactions of living cells. Enzymes are classified using a hierarchical numbering system called Enzyme Commission (EC) numbers that describe the chemical reactions the enzymes catalyze, from a general reaction type (e.g., breaking bonds, transferring chemical groups, etc.) to more specific aspects such as chemical bonds and substrates involved in the reaction. We present a new machine learning method for predicting EC numbers called Deep Enzyme Function Transfer (DEFT). This method improves on previous methods that use either protein sequence- or three-dimensional (3D) structure-based comparisons between enzymes of known and unknown classification. DEFT combines the strengths of both approaches by first using a protein sequence-based model to predict the general enzyme category and then using protein structure comparisons to predict the finer subcategories. We demonstrate that DEFT achieves superior accuracy compared with current state-of-the-art tools for EC number prediction. We next demonstrate how DEFTs computational efficiency enables us to perform high-throughput, genome-wide annotations of organisms enzyme repertoires. We illustrate this capability by experimentally validating DEFT predicted sugar metabolizing enzyme profiles of intestinal mucus associated bacteria.

bioinformatics↗

Computational design of blue melanin with peptide motif scaffolding

De novo melanin design seeks to extend natural melanin colors to new, stable colors (blue, purple, green) with sequence-to-color tunability. Natural melanin, polymerized from tyrosine (Y), is a robust pigment with heterogenous molecular weights. Control of melanin size (length) is challenging; thus, only specific colors (yellow to brown) exist in nature. In this work, we describe the design of blue melanin through the polymerization of Y-containing pentapeptides with two key properties: tight packing during peptide assembly and high solubility in aqueous environments. By motif scaffolding a pentapeptide-repeat protein (PRP) with RFdiffusion, we narrowed 160,000 possible combinations to a library of 905 Y-containing pentapeptides with tight packing features. Two of the most soluble designs successfully formed stable blue melanin with {lambda}max absorbing in 615-620 nm, contributed by homogeneous melanin length achieved around 60 Y units. Other designs also formed new colors (purple, green), along with more known colors (red, yellow, brown). We found that blue melanin exhibited thermal stability at an autoclave temperature of 121{degrees}C and photostability of weeks under 600 lux illumination. We also demonstrated the application of blue melanin as an electrophoretic ink. De novo color design from simple peptides could potentially transform how colorants are sourced and produced. Our approach with computational design should also inspire the development of new deep-learning tools to directly predict colors from amino acid sequences.

biochemistry↗

Synergetic hallmark knockouts immortalize bovine muscle stem cells for cellular agriculture

Achieving scalable and sustainable production of cultivated meat hinges on developing robust livestock muscle and fat cell lines that can proliferate and differentiate effectively, while meeting regulatory standards and consumer expectations. In this study, bovine satellite cells (BSCs) were immortalized using CRISPR/Cas9 to knockdown PTEN (phosphatase and tensin homolog), TP53 (cellular tumor antigen) and SMAD4 (SMAD [Sma and Mad proteins] family member 4). The resulting cell line, named "CriBSC2," exhibited consistent growth, maintained muscle cell characteristics, and successfully differentiated into multinucleated myotubes after more than 150 cell divisions. In contrast, another cell line, "CriBSC1," achieved immortalization with TP53 knockout alone but lacked differentiation capacity. CriBSC2 were further cultured on gelatin scaffolds to evaluate their anchorage-dependent responses, laying the groundwork for their potential application in tissue engineering for cellular agriculture. Ultimately, CriBSC2 cells demonstrate suitable proliferation and differentiation capabilities crucial for advancing cellular agriculture and future food technologies.

cell biology↗

Comparative Transcriptomics of Adherent and Suspension Chicken Fibroblast Cell Lines for the Optimization of Cultivated Meat Processes

For the cultivated meat industry, food-relevant cell lines with optimal attributes for industrial bioprocesses are needed. One key trait is suspension-proficiency, or the ability of cells to proliferate in non-adherent culture conditions, to achieve high viable cell densities and streamline cell harvests. The limited success with suspension adaptations of animal cells for cultivated meat restricts the number of non-adherent cell lines suitable for cost-efficient biomass production. Here, we investigated transcriptional profiles of chicken embryonic fibroblasts (DF-1s) as they progressed from adherent to suspension culture using time-series RNA sequencing. From this analysis, we identified that DF-1s adapt to their non-adherent conditions by suppressing non-essential metabolism and tightly regulating cell junctions in response to oxidative stress and cell detachment. Based on these findings, we propose a novel mechanism enabling suspension proficiency in DF-1s, where mitogen-activated protein kinase (MAPK) p38{delta} sequesters TEA domain (TEAD) proteins to the cytoplasm and promotes Yes-associated protein (YAP) binding to Forkhead box protein 1 (FoxO1), simultaneously causing cell-matrix dissociation and antioxidant production. The outcome of this work is a deeper understanding of mechanisms that drive the generation of suspension cell lines, with a goal to translate these findings to other food-relevant cell lines and develop expanded cell options for the field.

cell biology↗

Modulation of Nutritional Composition and Aroma Volatiles in Cultivated Pork Fat by Culture Media Supplementation

Cultivated meat is emerging as a novel food source with the potential to contribute to a more sustainable and ethical food production system. However, limited research to date has explored the extent to which the nutrition and the aroma of such foods can be altered through cell culture conditions. Here, we aimed to modulate the aromatic volatile compounds in heated porcine cultivated fat cells by manipulating the media components while ensuring the preservation of robust fat differentiation. Using dynamic headspace gas chromatography-mass spectrometry (DHS-GC-MS), we demonstrated that supplementing cells with thiamine-HCl increased its intracellular concentration and promoted the production of 4-methyl-5-thiazoleethanol, contributing to milky aroma. Similarly, supplementation with L-methionine enhanced its intracellular concentration and increased the production of methional, a volatile compound with a potato-like aroma. Additionally, myoglobin significantly altered the volatile organic compound profile of cultivated fat. Notably, the concentration of {gamma}-nonalactone, (E,E)-2,4-decadienal and 2-pentylfuran were increased, which contribute to a coconut-like, deep fat, fruity aroma, respectively, as well as elevated levels of other alcohols, aldehydes and furans. These findings highlight the potential of culture media formulations to modulate the aroma in cultivated fat production, a unique opportunity to optimize sensory features using this novel food production technology. HighlightsNutrient composition and aroma profiles of cultivated pork fat upon baking were modulated by cell culture media supplementation. Supplementing with thiamine-HCl, L-methionine, or myoglobin increased intracellular levels of thiamine or methionine and modulated the formation of aroma volatiles, enhancing characteristic odors such as milky, potato-like, and coconut-like notes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/667495v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@11cd7c4org.highwire.dtl.DTLVardef@99b290org.highwire.dtl.DTLVardef@9a47beorg.highwire.dtl.DTLVardef@dc1689_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical overview of the methodology. Porcine dedifferentiated fat cells (pDFAT) were differentiated into adipocytes using adipogenesis media supplemented with aroma precursors. The cells were heated (cooked) and the resulting volatile compounds were analyzed using dynamic headspace gas chromatography-mass spectrometry (DHS-GC-MS).

biochemistry↗

Microbial lysates as low-cost serum replacements in cellular agriculture media formulation

Cultivated meat, the process of generating meat in vitro without sacrificing animals, is a promising alternative to the traditional practice of livestock agriculture. However, the success of this field depends on finding sustainable and economical replacements for animal-derived and expensive fetal bovine serum (FBS) that is typically used in cell culture processes. Here, we outline an effective screening process to vet the suitability of microbial lysates to support the growth of immortalized bovine satellite cells (iBSCs) and mackerel (Mack1) cells. We show that easily producible, low-cost whole-cell lysates from Vibrio natriegens can be used to create serum-free media for the long-term growth of iBSCs. The optimized medium, named "VN40" (basal B8 media containing Vibrio natriegens lysate proteins at 40 {micro}g/mL), outperforms previously established serum-free media while maintaining cell phenotype and myogenicity. Overall, this study shows a novel approach to producing serum-free media for cultivated meat production using microbially-derived lysates.

bioengineering↗

Establishment & Characterization of a Non-Adherent Insect Cell Line for Cultivated Meat

This study presents a blueprint for developing, scaling, and analyzing novel insect cell lines for food. The large-scale production of cultivated meat requires the development and analysis of cell lines that are simple to grow and easy to scale. Insect cells may be a favorable cell source due to their robust growth properties, adaptability to different culture conditions, and resiliency in culture. Cells were isolated from Tobacco hornworm (Manduca sexta) embryos and subsequently adapted to single-cell suspension culture in animal-free growth media. Cells were able to reach relatively high cell densities of over 20 million cells per mL in shake flasks. Cell growth data is presented in various culture vessels and spent media analysis was performed to better understand cell metabolic processes. Finally, a preliminary nutritional profile consisting of proximate, amino acid, mineral, and fatty acid analysis is reported. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/618906v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@ef6bbborg.highwire.dtl.DTLVardef@89c37eorg.highwire.dtl.DTLVardef@81a024org.highwire.dtl.DTLVardef@38bc91_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

Multi-modal, Label-free, Optical Mapping of Cellular Metabolic Function and Oxidative Stress in 3D Engineered Brain Tissue Models

Brain metabolism is essential for the function of organisms. While established imaging methods provide valuable insights into brain metabolic function, they lack the resolution to capture important metabolic interactions and heterogeneity at the cellular level. Label-free, two-photon excited fluorescence imaging addresses this issue by enabling dynamic metabolic assessments at the single-cell level without manipulations. In this study, we demonstrate the impact of spectral imaging on the development of rigorous intensity and lifetime label-free imaging protocols to assess dynamically metabolic functions over time in 3D engineered brain tissue models comprised of human induced neural stem cells, astrocytes, and microglia. Specifically, we rely on multi-wavelength spectral imaging to identify the excitation/emission profiles of key cellular fluorophores within human brain cells, including NAD(P)H, LipDH, FAD, and lipofuscin. These enable the development of methods to mitigate lipofuscins overlap with NAD(P)H and flavin autofluorescence to extract reliable optical metabolic function metrics from images acquired at two excitation wavelengths over two emission bands. We present fluorescence intensity and lifetime metrics reporting on redox state, mitochondrial fragmentation, and NAD(P)H binding status in neuronal monoculture and the triculture systems to highlight the functional impact of metabolic interactions between different cell types. Our findings reveal significant metabolic differences between neurons and glial cells, shedding light on metabolic pathway utilization, including the glutathione pathway, OXPHOS, glycolysis, and fatty acid oxidation. Collectively, our studies establish a label-free, non-destructive approach to assess the metabolic function and interactions among different brain cell types relying on endogenous fluorescence and illustrate the complementary nature of the information that is gained by combining intensity and lifetime-based images. Such methods can improve understanding of physiological brain function and dysfunction that occurs at the onset of cancers, traumatic injuries and neurodegenerative diseases.

bioengineering↗

Chemical and Sensory Analyses of Cultivated Pork Fat Tissue as a Flavor Enhancer for Meat Alternatives

The emerging field of cellular agriculture has accelerated the development of cell-cultivated adipose tissue as an additive to enhance the flavor of alternative meat products. However, there has been limited research to evaluate the sensory profile of in vitro-grown tissues compared to conventionally obtained animal fat. This study aimed to investigate the aromatic characteristics of cell-cultivated fat tissue as a flavor enhancer for meat alternatives. Porcine dedifferentiated fat cells were clonally isolated and differentiated into adipocytes. This cultured adipose tissue was then analyzed alongside native porcine fat using gas chromatography-mass spectrometry (GC/MS) coupled with descriptive sensory analysis by human panelists. This evaluation enabled quantitative and qualitative assessments of volatile compounds released during cooking for both in vitro and in vivo porcine fats. The volatile profiles generated during the cooking process and fatty aroma characteristics reported by sensory panelists were largely similar between the two fat sources, with some differences in the concentration of select compounds and aroma attributes. Ultimately, the panelists found comparable overall liking scores reported between the conventional and cultured porcine fats. These findings provide valuable sensory evidence supporting the viability of cell-cultivated adipose tissue as a flavor component of meat alternatives, substituting for conventional animal fat.

bioengineering↗

Decellularized Banana Leaves: Eco-Friendly Scaffolds for Cell-Based Seafood

Cellular agriculture, as an emerging food production system, holds potential to address sustainability, food security, and agricultural resilience. Within the cell-based meat supply chain, one of the key steps is scaffolding. In this study, we assessed decellularized banana leaves, various coating materials, and different cell seeding strategies to determine their effects on cell viability, cell growth, cell alignment, and the response of the materials to thermal processing. The efficiency of decellularization was verified through DNA quantification, which decreased from 445 ng/mg in fresh banana leaves to non-detectable levels in the decellularized samples. This was further confirmed by FTIR and PCA modeling. Cell viability exceeded 98% on uncoated, soy-coated, and gelatin-coated samples of the decellularized banana leaves. Alignment of cells on gelatin-coated samples was the highest among the samples, with a dominant orientation of 65.8{degrees}, compared to soy-coated and uncoated samples with dominant orientations of 9.2{degrees} and -6.3{degrees}, respectively. In terms of quality attributes, the kinetics of shrinkage indicated that coating with soy and the presence of cells increased the activation energy due to the higher energy required for protein denaturation. Moreover, the kinetics of area changes in plain scaffolds without cells followed a first-order pattern, while with seeded cells a second-order pattern was followed. In summary, decellularized banana leaves present a sustainable and suitable biomaterial to support cells towards future needs related to meat production.

synthetic biology↗

Engineered autocrine signaling eliminates muscle cell FGF2 requirements for cultured meat production

Cultured meat is a promising technology that faces substantial cost barriers which are currently driven largely by the price of media components. Growth factors such as fibroblast growth factor 2 (FGF2) drive the cost of serum-free media for relevant cells including muscle satellite cells. Here, we engineered immortalized bovine satellite cells (iBSCs) for inducible expression of FGF2 and/or mutated RasG12V in order to overcome media growth factor requirements through autocrine signaling. Engineered cells were able to proliferate over multiple passages in FGF2-free medium, thereby eliminating the need for this costly component. Additionally, cells maintained their myogenicity, albeit with reduced differentiation capacity. Ultimately, this offers a proof-of-principle for lower-cost cultured meat production through cell line engineering.

bioengineering↗

Immortalized bovine satellite cells for cultured meat applications

For cultured meat to succeed at scale, muscle cells from food-relevant species must be expanded in vitro in a rapid and reliable manner to produce millions of metric tons of biomass annually. Toward this goal, genetically immortalized cells offer substantial benefits over primary cells, including rapid growth, escape from cellular senescence, and consistent starting cell populations for production. Here, we develop genetically immortalized bovine satellite cells (iBSCs) via constitutive expression of bovine Telomerase reverse transcriptase (TERT) and Cyclin-dependent kinase 4 (CDK4). These cells achieve over 120 doublings at the time of publication and maintain their capacity for myogenic differentiation. They therefore offer a valuable tool to the field, enabling further research and development to advance cultured meat.

cell biology↗

Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury, composed of neurons, microglia, and astrocytes, and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for the development of therapies for TBI patients.

neuroscience↗