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At least 1,135 records · Page 63

Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane‐associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

Springer, David L.↗

Structure of a Rhamnogalacturonan Fragment from Apple Pectin: Implications for Pectin Architecture

A commercial apple pectin was sequentially digested with the cloned enzymes endopolygalacturonase, galactanase, arabinofuranosidase, xylogalacturonase, and rhamnogalacturonan hydrolase. The rhamnogalacturonan hydrolase-generated oligosaccharides were separated by ultrafiltration, anion exchange, and size-exclusion chromatography. Fractions from the ion exchange chromatography were pooled, lyophilized, and screened by MALDI-TOF MS. An oligosaccharide (RGP14P3) was identified and its structure, α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A- ( 1 → 2 ) - α -L-Rha p - ( 1 → 4 ) - α -D-Gal p A, determined by 1D and 2D NMR spectrometry. This oligosaccharide probably represents a direct connection between homogalacturonan and rhamnogalacturonan in pectin. Alternatively, it could indicate that the nonreducing end of rhamnogalacturonan starts with a galacturonic acid residue.

Wu, Xiangmei↗

Oak Ridge Graph Analytics for Medical Innovation (ORiGAMI)

In this era of data-driven decisions and discovery where Big Data is producing Bigger Data, data scientists at the Oak Ridge National Laboratory are leveraging unique leadership infrastructure (e.g., Urika XA and Urika GD appliances) to develop scalable algorithms for semantic, logical and statistical reasoning with Big Data (i.e., data stored in databases as well as unstructured data in documents). ORiGAMI is a next-generation knowledge-discovery framework that is: (a) knowledge nurturing (i.e., evolves seamlessly with newer knowledge and data), (b) smart and curious (i.e. using information-foraging and reasoning algorithms to digest content) and (c) synergistic (i.e., interfaces computers with what they do best to help subject-matter-experts do their best. ORiGAMI has been demonstrated using the National Library of Medicine's SEMANTIC MEDLINE (archive of medical knowledge since 1994).

Roberts, LarryW.↗

Crosslinking IMS

Given a list of proteins, finds all cross-linked peptides in the given Ion Mobility MS dataset. This application attempts to find all cross-linked peptides in the given Ion Mobility MS dataset. First, the FASTA file is read in and possible peptides are generated by digesting the protein(s) and assuming 1 max missed cleavage. The possible peptides are searched for in the given features by matching up the monoisotopic mass of the feature to the mass of the peptides. The mass ppm tolerance is an input parameter of the application. All possible cross-links of the unmodified peptides are then calculated and searched for in the Isotopic Peak data. A detailed csv results file is created at the end.

Monroe, Matthew↗

Smelly-Odor Webtool (Smelly) v1

Smelly is an interactive webtool displaying odor dispersion from various potential waste-to-energy sites across California. It currently displays data for dry anaerobic digestion technology with organic waste intake of 90,000 tons per year. The model addresses meteorological and geographical differences in offsite odor impacts (Jin et al. 2019). In addition to interactive display of odor dispersions, the embedded emission calculator provides two additional functionalities: (1) determine the impact distance for given onsite odor emission rates; and (2) determine allowable odor emission rates at given separation distance between the facility and its downwind communities. Please note that data and model is preliminary at this point and serve as a Proof of Concept. Additional data collection, modeling and research is needed for final application.

Jin, Ling↗

BioSiting Tool (BioSiting) v2

The BioSiting Tool provides a geospatial interface for analyzing bioeconomy resources and infrastructure across the continental U.S. The tool integrates empirical and modeled data from a broad range of sources. Bioeconomy resources mapped in the tool include agricultural residues, forest residues, municipal solid waste streams, food waste, manure, fats, oils and greases and potential yields of energy crops. Infrastructure mapped in the tool includes biorefineries, material recovery facilities, anaerobic digesters, wastewater treatment plants, combustion plants, district energy systems, crude oil pipelines, petroleum pipelines, natural gas pipelines, railways and freight terminals. Additional data layers include environmental justice indicators at the census tract level and carbon dioxide geologic storage potential. Users can select a location on the map, define a buffer radius in kilometers and generate an inventory of all bioecomony resources within the buffer zone. Data from the tool can be downloaded from individual buffer zones, or at the state or national level.

Huntington, Tyler↗

EMSL-Computing/pspecterlib

Proteomics R package for matching peptide fragments for both digested and intact proteomics

Degnan, David [Pacific Northwest National Laborato↗

First PET Imaging Studies With 63 Zn-Zinc Citrate in Healthy Human Participants and Patients With Alzheimer Disease

Abnormalities in zinc homeostasis are indicated in many human diseases, including Alzheimer disease (AD). 63 Zn-zinc citrate was developed as a positron emission tomography (PET) imaging probe of zinc transport and used in a first-in-human study in 6 healthy elderly individuals and 6 patients with clinically confirmed AD. A dynamic PET imaging of the brain was performed for 30 minutes following intravenous administration of 63 Zn-zinc citrate (~330 MBq). Subsequently, body PET images were acquired. Urine and venous blood were analyzed to give information on urinary excretion and pharmacokinetics. Regional cerebral 63 Zn clearances were compared with 11 C-Pittsburgh Compound B ( 11 C-PiB) and 18 F-fluorodeoxyglucose ( 18 F-FDG) imaging data. 63 Zn-zinc citrate was well tolerated in human participants with no adverse events monitored. Tissues of highest uptake were liver, pancreas, and kidney, with moderate uptake being seen in intestines, prostate (in males), thyroid, spleen, stomach, pituitary, and salivary glands. Moderate brain uptake was observed, and regional dependencies were observed in 63 Zn clearance kinetics in relationship with regions of high amyloid-β plaque burden ( 11 C-PiB) and 18 F-FDG hypometabolism. In conclusion, zinc transport was successfully imaged in human participants using the PET probe 63 Zn-zinc citrate. Primary sites of uptake in the digestive system accent the role of zinc in gastrointestinal function. Preliminary information on zinc kinetics in patients with AD evidenced regional differences in clearance rates in correspondence with regional amyloid-β pathology, warranting further imaging studies of zinc homeostasis in patients with AD.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis

Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro . The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. We used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7 . A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro . Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.

09 BIOMASS FUELS↗

A coarse-grained model for synergistic action of multiple enzymes on cellulose

In this study, degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing -1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, -glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose, several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. As a result, we present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages) and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. In conclusion, our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures the endo-exo synergism of cellulase enzyme cocktails. This model constitutes a critical step towards testing hypotheses and understanding approaches for maximizing synergy and substrate properties with a goal of cost effective enzymatic hydrolysis.

59 BASIC BIOLOGICAL SCIENCES↗

Impacts of Deacetylation Prior to Dilute Acid Pretreatment on the Bioethanol Process

Dilute acid pretreatment is a promising pretreatment technology for the biochemical production of ethanol from lignocellulosic biomass. During dilute acid pretreatment, xylan depolymerizes to form soluble xylose monomers and oligomers. Because the xylan found in nature is highly acetylated, the formation of xylose monomers requires two steps: (1) cleavage of the xylosidic bonds, and (2) cleavage of covalently bonded acetyl ester groups. Results: In this study, we show that the latter may be the rate limiting step for xylose monomer formation. Furthermore, acetyl groups are also found to be a cause of biomass recalcitrance and hydrolyzate toxicity. While the removal of acetyl groups from native corn stover by alkaline de-esterification prior to pretreatment improves overall process yields, the exact impact is highly dependent on the corn stover variety in use. Xylose monomer yields in pretreatment generally increases by greater than 10%. Compared to pretreated corn stover controls, the deacetylated corn stover feedstock is approximately 20% more digestible after pretreatment. Finally, by lowering hydrolyzate toxicity, xylose utilization and ethanol yields are further improved during fermentation by roughly 10% and 7%, respectively. In this study, several varieties of corn stover lots were investigated to test the robustness of the deacetylation-pretreatment-saccharification-fermentation process. Conclusions: Deacetylation shows significant improvement on glucose and xylose yields during pretreatment and enzymatic hydrolysis, but it also reduces hydrolyzate toxicity during fermentation, thereby improving ethanol yields and titer. The magnitude of effect is dependent on the selected corn stover variety, with several varieties achieving improvements of greater than 10% xylose yield in pretreatment, 20% glucose yield in low solids enzymatic hydrolysis and 7% overall ethanol yield.

09 BIOMASS FUELS↗

Life-cycle energy use and greenhouse gas emissions of production of bioethanol from sorghum in the United States

Background: The availability of feedstock options is a key to meeting the volumetric requirement of 136.3 billion liters of renewable fuels per year beginning in 2022, as required in the US 2007 Energy Independence and Security Act. Life-cycle greenhouse gas (GHG) emissions of sorghum-based ethanol need to be assessed for sorghum to play a role in meeting that requirement. Results: Multiple sorghum-based ethanol production pathways show diverse well-to-wheels (WTW) energy use and GHG emissions due to differences in energy use and fertilizer use intensity associated with sorghum growth and differences in the ethanol conversion processes. All sorghum-based ethanol pathways can achieve significant fossil energy savings. Relative to GHG emissions from conventional gasoline, grain sorghum-based ethanol can reduce WTW GHG emissions by 35% or 23%, respectively, when wet or dried distillers grains with solubles (DGS) is the co-product and fossil natural gas (FNG) is consumed as the process fuel. The reduction increased to 56% or 55%, respectively, for wet or dried DGS co-production when renewable natural gas (RNG) from anaerobic digestion of animal waste is used as the process fuel. These results do not include land-use change (LUC) GHG emissions, which we take as negligible. If LUC GHG emissions for grain sorghum ethanol as estimated by the US Environmental Protection Agency (EPA) are included (26 g CO2e/MJ), these reductions when wet DGS is co-produced decrease to 7% or 29% when FNG or RNG is used as the process fuel. Sweet sorghum-based ethanol can reduce GHG emissions by 71% or 72% without or with use of co-produced vinasse as farm fertilizer, respectively, in ethanol plants using only sugar juice to produce ethanol. If both sugar and cellulosic bagasse were used in the future for ethanol production, an ethanol plant with a combined heat and power (CHP) system that supplies all process energy can achieve a GHG emission reduction of 70% or 72%, respectively, without or with vinasse fertigation. Forage sorghum-based ethanol can achieve a 49% WTW GHG emission reduction when ethanol plants meet process energy demands with CHP. In the case of forage sorghum and an integrated sweet sorghum pathway, the use of a portion of feedstock to fuel CHP systems significantly reduces fossil fuel consumption and GHG emissions. Conclusions: This study provides new insight into life-cycle energy use and GHG emissions of multiple sorghum-based ethanol production pathways in the US. Our results show that adding sorghum feedstocks to the existing options for ethanol production could help in meeting the requirements for volumes of renewable, advanced and cellulosic bioethanol production in the US required by the EPA’s Renewable Fuel Standard program.

09 BIOMASS FUELS↗

Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates

Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. Method. We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

09 BIOMASS FUELS↗

Identification of carbohydrate gene clusters obtained from in vitro fermentations as predictive biomarkers of prebiotic responses

Prebiotic fibers are non-digestible substrates that modulate the gut microbiome by promoting expansion of microbes having the genetic and physiological potential to utilize those molecules. Although several prebiotic substrates have been consistently shown to provide health benefits in human clinical trials, responder and non-responder phenotypes are often reported. These observations had led to interest in identifying, a priori, prebiotic responders and non-responders as a basis for personalized nutrition. In this study, we conducted in vitro fecal enrichments and applied shotgun metagenomics and machine learning tools to identify microbial gene signatures from adult subjects that could be used to predict prebiotic responders and non-responders. Using short chain fatty acids as a targeted response, we identified genetic features, consisting of carbohydrate active enzymes, transcription factors and sugar transporters, from metagenomic sequencing of in vitro fermentations for three prebiotic substrates: xylooligosacharides, fructooligosacharides, and inulin. A machine learning approach was then used to select substrate-specific gene signatures as predictive features. These features were found to be predictive for XOS responders with respect to SCFA production in an in vivo trial. Our results confirm the bifidogenic effect of commonly used prebiotic substrates along with inter-individual microbial responses towards these substrates. We successfully trained classifiers for the prediction of prebiotic responders towards XOS and inulin with robust accuracy (≥ AUC 0.9) and demonstrated its utility in a human feeding trial. Overall, the findings from this study highlight the practical implementation of pre-intervention targeted profiling of individual microbiomes to stratify responders and non-responders.

59 BASIC BIOLOGICAL SCIENCES↗

Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification range from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present it is unknown if this also affects the activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.

59 BASIC BIOLOGICAL SCIENCES↗

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalisgenerates a new host for the analysis of biomass deconstruction

Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria. To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host. The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.

09 BIOMASS FUELS↗

Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry

For the development of lignocellulosic biofuels a common strategy to release hemicellulosic sugars and enhance the enzymatic digestibility of cellulose is the heat pretreatment of biomass with dilute acid. During this process, fermentation inhibitors such as 5-hydroxymethylfurfural, furfural, phenolics, and organic acids are formed and released into the so-called hydrolysate. The phenolic inhibitors have been studied fairly extensively, but fewer studies have focused on the analysis of the organic acids profile. For this purpose, a simple and fast liquid chromatography/mass spectrometry (LC/MS) method for the analysis of organic acids in the hydrolysate has been developed using an ion exchange column based on a polystyrene-divinylbenzene polymer frequently used in biofuel research. The application of the LC/MS method to a hydrolysate from Miscanthus has been evaluated.

Biotechnology & Applied Microbiology↗

Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance

A major obstacle, and perhaps the most important economic barrier to the effective use of plant biomass for the production of fuels, chemicals, and bioproducts, is our current lack of knowledge of how to efficiently and effectively deconstruct wall polymers for their subsequent use as feedstocks. Plants represent the most desired source of renewable energy and hydrocarbons because they fix CO 2 , making their use carbon neutral. Their biomass structure, however, is a barrier to deconstruction, and this is often referred to as recalcitrance. Members of the bacterial genus Caldicellulosiruptor have the ability to grow on unpretreated plant biomass and thus provide an assay for plant deconstruction and biomass recalcitrance. Using recently developed genetic tools for manipulation of these bacteria, a deletion of a gene cluster encoding enzymes for pectin degradation was constructed, and the resulting mutant was reduced in its ability to grow on both dicot and grass biomass, but not on soluble sugars. The plant biomass from three phylogenetically diverse plants, Arabidopsis (a herbaceous dicot), switchgrass (a monocot grass), and poplar (a woody dicot), was used in these analyses. These biomass types have cell walls that are significantly different from each other in both structure and composition. While pectin is a relatively minor component of the grass and woody dicot substrates, the reduced growth of the mutant on all three biomass types provides direct evidence that pectin plays an important role in biomass recalcitrance. Glycome profiling of the plant material remaining after growth of the mutant on Arabidopsis biomass compared to the wild-type revealed differences in the rhamnogalacturonan I, homogalacturonan, arabinogalactan, and xylan profiles. In contrast, only minor differences were observed in the glycome profiles of the switchgrass and poplar biomass. In conclusion, the combination of microbial digestion and plant biomass analysis provides a new and important platform to identify plant wall structures whose presence reduces the ability of microbes to deconstruct plant walls and to identify enzymes that specifically deconstruct those structures.

60 APPLIED LIFE SCIENCES↗