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At least 559 records · Page 31

Tuning gold-based surface functionalization for streptavidin detection: A combined simulative and experimental study

A rationally designed gold-functionalized surface capable of capturing a target protein is presented using the biotin–streptavidin pair as a proof-of-concept. We carried out multiscale simulations to shed light on the binding mechanism of streptavidin on four differently biotinylated surfaces. Brownian Dynamics simulations were used to reveal the preferred initial orientation of streptavidin over the surfaces, whereas classical molecular dynamics was used to refine the binding poses and to investigate the fundamental forces involved in binding, and the binding kinetics. We assessed the binding events and the stability of the streptavidin attachment through a quartz crystal microbalance with dissipation monitoring (QCM-D). The sensing element comprises of biotinylated polyethylene glycol chains grafted on the sensor’s gold surface via thiol-Au chemistry. Finally, we compared the results from experiments and simulations. We found that the confined biotin moieties can specifically capture streptavidin from the liquid phase and provide guidelines on how to exploit the microscopic parameters obtained from simulations to guide the design of further biosensors with enhanced sensitivity.

59 BASIC BIOLOGICAL SCIENCES↗

The Measurement of Reversible Redox Dependent Post-translational Modifications and Their Regulation of Mitochondrial and Skeletal Muscle Function

Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar and excitation-contraction (EC) coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.

Environmental Molecular Sciences Laboratory↗

Bimodal effect of hydrogen peroxide and oxidative events in nitrite-induced rapid root abscission by the water fern Azolla pinnata

In the genus Azolla rapid abscission of roots from floating fronds occurs within minutes in response to a variety of stresses, including exposure to nitrite. We found that hydrogen peroxide, though itself not an inducer of root abscission, modulates nitrite-induced root abscission by Azolla pinnata in a dose-dependent manner, with 2 mM H 2 O 2 significantly diminishing the responsiveness to 2 mM NaNO 2 , and 10 mM H 2 O 2 slightly enhancing it. Hypoxia, which has been found in other plants to result in autogenic production of H 2 O 2 , dramatically stimulated root abscission of A. pinnata in response to nitrite, especially for plants previously cultivated in medium containing 5 mM KNO 3 compared to plants cultivated under N 2 -fixing conditions without combined nitrogen. Plants, including Azolla , produce the small signaling molecule nitric oxide (NO) from nitrite using nitrate reductase. We found Azolla plants to display dose-dependent root abscission in response to the NO donor spermine NONOate. Treatment of plants with the thiol-modifying agents S-methyl methanethiosulfonate or glutathione inhibited the nitrite-induced root abscission response. Synchrotron radiation-based Fourier transform infrared spectromicroscopy revealed higher levels of carbonylation in the abscission zone of dropped roots, indicative of reaction products of polysaccharides with potent free radical oxidants. Lastly, we hypothesize that metabolic products of nitrite and NO react with H 2 O 2 in the apoplast leading to free-radical-mediated cleavage of structural polysaccharides and consequent rapid root abscission.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Examining Interactions of Uranyl(VI) Ions with Amino Acids in the Gas Phase

Gas-phase experiments, using electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT/MS), were conducted to probe basic interactions of the uranyl(VI) ion, UO 2 2+ , with selected natural amino acids, namely, L-cysteine (Cys), L-histidine (His), and L-aspartic acid (Asp), which strongly bind to metal ions. The simplest amino acid, glycine (Gly), was also studied for comparison. Cys, His, and Asp have additional potentially coordinating groups beyond the amino and carboxylic acid functional groups, specifically thiol in Cys, imidazole in His, and a second carboxylate in Asp. Gas-phase experiments comprised collision-induced dissociation (CID) of uranyl–amino acid complexes and competitive CID to assess the relative binding strength of different amino acids in the same uranyl complex. Reactivity of selected uranyl–amino acid complexes with water provided further insights into relative stabilities. In positive ion mode, CID and ensuing reactions with water suggested that uranyl–neutral AA binding strength decreased in the order His > Asp > Cys > Gly, which is similar to amino acid proton affinities. In negative ion mode, CID revealed a decreasing dissociation tendency in the order Gly >> His ≈ Cys > Asp, presumably reflecting a reverse enhanced binding to uranyl of the doubly deprotonated amino acids formed in CID.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

Structural Insights into Mechanisms Underlying Mitochondrial and Bacterial Cytochrome c Synthases

Mitochondrial holocytochrome c synthase (HCCS) is an essential protein in assembling cytochrome c (cyt c) of the electron transport system. HCCS binds heme and covalently attaches the two vinyls of heme to two cysteine thiols of the cyt c CXXCH motif. Human HCCS recognizes both cyt c and cytochrome c1 of complex III (cytochrome bc1). HCCS is mutated in some human diseases and it has been investigated recombinantly by mutational, biochemical, and reconstitution studies in the past decade. Here, we employ structural prediction programs (e.g., AlphaFold 3) on HCCS and its two substrates, heme and cytochrome c. The results, when combined with spectroscopic and functional analyses of HCCS and variants, provide insights into the structural basis for heme binding, apocyt c binding, covalent attachment, and release of the holocyt c product. Results from in vitro reconstitution of purified human HCCS using cyt c and cyt c1 peptides as acceptors are consistent with the structural modeling of substrate binding. Reconstitution of HCCS and cyt c1 provides an approach to studying cyt c1 assembly, which has been refractile to recombinant in vivo reconstitution (unlike HCCS and cyt c). We propose a structural basis for release of the holocyt c product from HCCS based on in vitro studies and on cryoEM structures of the bacterial cyt c synthase (CcsBA) active site. We analyze the kinetoplastid mitochondrial synthase (KCCS), and hypothesize a molecular evolutionary path from mitochondrial endosymbiosis to the current HCCS.

Biochemistry & Molecular Biology↗

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin-like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.

59 BASIC BIOLOGICAL SCIENCES↗

Stability of Polyethylene Glycol-Coated Copper Nanoparticles and Their Optical Properties

Oxidation is a corrosion reaction where the corroded metal forms an oxide. Prevention of oxidation at the nanoscale is critically important to retain the physicochemical properties of metal nanoparticles. In this work, we studied the stability of polyethylene glycol (PEG) coated copper nanoparticles (PEGylated CuNPs) against oxidation. The freshly-prepared PEGylated CuNPs mainly consist of metallic Cu which are quite stable in air although their surfaces are typically covered with a few monolayers of cuprous oxide. However, they are quickly oxidized in water due to the presence of protons that facilitate oxidation of the cuprous oxide to cupric oxide. PEG with carboxylic acid terminus could slightly delay the oxidation process compared to that with thiol terminus. It was found that a solvent with reducing power such as ethanol could greatly enhance the stability of PEGylated CuNPs by preventing further oxidation of the cuprous oxide to cupric oxide and thus retain the optical properties of CuNPs. The reducing environment also assists the galvanic replacement of these PEGylated CuNPs to form hollow nanoshells; however, they consist of ultra-small particle assemblies due to the co-reduction of gold precursor during the replacement reaction. As a result, these nanoshells do not exhibit strong optical properties in the near-infrared region. This study highlights the importance of solvent effects on PEGylated nonprecious metal nanoparticles against oxidation corrosion and its applications in preserving physicochemical properties of metallic nanostructures.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Synthesis of Terpyridine-Terminated Amphiphilic Block Copolymers and Their Self-Assembly into Metallo-Polymer Nanovesicles

Polystyrene-b-polyethylene glycol (PS-b-PEG) amphiphilic block copolymers featuring a terminal tridentate N,N,N-ligand (terpyridine) were synthesized for the first time through an efficient route. In this approach, telechelic chain-end modified polystyrenes were produced via reversible addition-fragmentation chain-transfer (RAFT) polymerization by using terpyridine trithiocarbonate as the chain-transfer agent, after which the hydrophilic polyethylene glycol (PEG) block was incorporated into the hydrophobic polystyrene (PS) block in high yields via a thiol-ene process. Following metal-coordination with Mn2+, Fe2+, Ni2+, and Zn2+, the resulting metallo-polymers were self-assembled into spherical, vesicular nanostructures, as characterized by dynamic light scattering and transmission electron microscopy (TEM) imaging.

36 MATERIALS SCIENCE↗

Selected Area Deposition of High Purity Gold for Functional 3D Architectures

Selected area deposition of high purity gold films onto nanoscale 3D architectures is highly desirable as gold is conductive, inert, plasmonically active, and can be functionalized with thiol chemistries, which are useful in many biological applications. Here, we show that high-purity gold coatings can be selectively grown with the Me 2 Au (acac) precursor onto nanoscale 3D architectures via a pulsed laser pyrolytic chemical vapor deposition process. The selected area of deposition is achieved due to the high thermal resistance of the nanoscale geometries. Focused electron beam induced deposits (FEBID) and carbon nanofibers are functionalized with gold coatings, and we demonstrate the effects that laser irradiance, pulse width, and precursor pressure have on the growth rate. Furthermore, we demonstrate selected area deposition with a feature-targeting resolutions of ~100 and 5 µm, using diode lasers coupled to a multimode (915 nm) and single mode (785 nm) fiber optic, respectively. The experimental results are rationalized via finite element thermal modeling.

3-dimensional nanostructures↗

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

Here in this work, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 μm). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Lastly, detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

36 MATERIALS SCIENCE↗

Retrospective on Recent DOE-Funded Studies Concerning the Extraction of Rare Earth Elements & Lithium from Geothermal Brines (Final Report)

Rare earth elements (REE) and lithium are non-toxic metals that are considered critical materials due to their use in electronics, magnets, batteries, and a wide variety of industrial processes important for the economy and military preparedness. Demand for REE and lithium is increasing and these critical materials are imported, so identifying and exploiting domestic sources of REE and lithium is a national priority. The U.S. Department of Energy (DOE) Geothermal Technologies Office (GTO) has been in the forefront of sponsoring research investigating the potential recovery of REE, lithium, and other critical minerals from geothermal brines. It has been proposed that the future of geothermal energy should include “hybrid systems” that combine electricity generation with other revenue-generating activities, such as recovery of valuable and critical minerals, including REE and lithium. Two recent GTO funding opportunities have focused on the recovery of REE and other valuable minerals from geothermal brines. The research supported by the GTO’s mineral recovery program is focused on three areas: resource characterization, technology for the extraction of REE, and technology for the extraction of lithium (Tables 1 and 2). This report is a retrospective study examining the outcome of GTO’s two recent mineral recovery programs (DE-FOA-0001016 in FY 2014 and DE-FOA-0001376 in FY 2016). In this report, the knowledge, technology, and techniques that were developed by researchers funded by GTO are summarized and discussed. Four projects were funded to assess the concentrations and amounts of REE found in geothermal brines and oil field produced waters. The GTO-funded studies compiled publically available data on REE concentrations from brines and produced water from all over the USA. In addition, new samples were collected and characterized from major geothermal and hydrocarbon basins in the Western USA. The studies examined the relationship between lithology and REE concentrations and developed models examining the influence of geology on REE concentrations in produced brines. It was determined that REE are frequently found at higher concentrations in oil field produced water than geothermal brines, but that some geothermal areas had significant REE resources. Significant reservoirs of REE were identified in the Western USA. In some cases, concentrations of REE were more than 1000 times the concentrations found in seawater. Collectively, these studies represent a comprehensive picture of REE resources associated with geothermal and hydrocarbon systems in the USA. The studies did not examine lithium resources, but in some cases, lithium concentration data was collected. Data from these studies are housed in the Geothermal Data Repository (GDR) and represent a significant information resource and it is recommended that these data be further analyzed in a future study. Eight projects were funded to develop new technology for REE extraction from geothermal fluids. These projects investigated sorption as an approach for removal and recovery of REE from geothermal brines. The projects investigated cutting-edge technology for selective sorption of ions from complex solutions, including the application of metal-organic frameworks and biosorbent proteins. The REE sorption studies tested different combinations of metal-binding ligands and solid supports. The most promising metal-binding ligands for REE included phosphonic acid, thiol, and carboxylic acid functional groups. Ligands were attached or incorporated into a wide variety of solid supports. In most cases, attachment was via covalent bonding to organic resins, polymers, or silica-based supports. Most of the REE projects were conducted at a low technology readiness level (TRL) and showed promise, but direct comparison between technologies was not possible based on the available information. It is recommended that testing and reporting be standardized to the extent possible to facilitate comparisons between technologies. Two projects were directed at novel lithium extraction technology. Both projects investigated the use of inorganic sorbents, including manganese oxides. One study also examined the use of metal- ion imprinted polymers as selective ion-exchange resins for the separation of lithium and manganese from brines. Both approaches showed promise for the selective extraction of lithium from brines, including potentially geothermal brines. Results from these GTO studies indicated that selective REE and lithium extraction is possible, but interference from co-occurring solutes, such as calcium, magnesium, or heavy metals, will interfere with process efficiency and negatively impact process economics. Techno-economic analysis conducted as part of the resource and technology studies suggest extraction of REE from geothermal brines is unlikely to be economically viable, especially since non-geothermal produced waters frequently have higher REE concentrations. It is recommended that benchmarks for techno-economic analysis be established to the extent possible for future studies, to facilitate direct comparison of various technologies. Based on the collective results of this program, it appears that hybrid geothermal power would benefit more from recovery of lithium and other metals, rather than REE. It is recommended that future studies be conducted at a higher- TRL and that sorbents be tested against actual geothermal fluid samples. Prior higher-TRL efforts to extract metals from geothermal brines should be further evaluated for lessons learned.

36 MATERIALS SCIENCE↗

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

59 BASIC BIOLOGICAL SCIENCES↗

Oxygen and sulfur interactions with a clean iron surface and the effect of rubbing contact on these interactions

An investigation was conducted to determine the nature of the chemical interactions of oxygen and sulfur with iron when surfaces are stationary and also during sliding in a vacuum environment. Various gases that contained sulfur, oxygen, or both were adsorbed to iron at 23 C. The gases included oxygen, hydrogen sulfide, methyl mercaptan, and sulfur dioxide. Friction experiments were conducted with a hemispherical rider sliding on a rotating disk. An Auger cylindrical mirror analyzer was used to monitor the iron surface chemistry. The results of this study indicate that oxygen will completely displace sulfide films from iron surfaces. Organic thiols containing sulfur, such as methyl mercaptan, adsorb to an iron surface dissociatively. Only sulfur is detected on the iron surface. Sliding inhibits the formation of sulfide films on iron with the adsorption of hydrogen sulfide and methyl mercaptan. With oxygen and sulfur dioxide, the sliding process does not affect adsorption behavior.

Buckley, D. H.↗

Raman spectra of gases. XVI - Torsional transitions in ethanol and ethanethiol

The Raman spectra of gaseous ethanol and ethanethiol have been investigated. Thiol torsional fundamentals for the gauche conformer of EtSH and EtSD have been observed and the asymmetric potential function for this vibration has been calculated. Methyl torsional transitions and overtones have also been observed for both of these molecules. Barriers to internal rotation for the methyl top are calculated to be 3.77 and 3.84 kcal/mol for the EtSH and EtSD compounds, respectively. Hydroxyl torsional fundamentals were observed at 207 and 170 per cm in the EtOH and EtOD spectra, respectively. Overtones of the methyl torsion in both molecules yield a barrier to internal rotation of 3.62 kcal/mol for the gauche conformer.

Durig, J. R.↗

Studies of the mechanisms of turbine fuel instability

The formation of insoluble deposits in a Jet A, a Diesel, and a model fuel (1/10 v/v tetralin/dodecane) was studied. Experiments were conducted using glass containers at 394 K with an air/fuel ratio of 14/1. The effects of addition of ppm levels of various compounds on deposit formation were evaluated. Nitrogen heterocycles were shown to produce a basicity dependent acceleration of deposition. Thiols and thiophene were shown to increase deposition while sulfides and disulfides act as inhibitors. Copper metal and its salts also promote deposition. Results of various instrumental analyses of deposits and development of a high performance liquid chromatographic method for monitoring deposit precursors are discussed.

Daniel, S. R.↗

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

Sulzner, Michael↗

A preliminary study of the variability and magnitude of the flux of biogenic sulfur gases from a New Hampshire salt marsh

Salt marshes have highly variable spatial and temporal fluxes of hydrogen sulfide, carbonyl sulfide, methane thiol, dimethyl sulfide, and carbon disulfide (H2S, COS, MeSH, DMS, and CS2, respectively). This variability was tested at nine emission sites in a New Hampshire, USA salt marsh: three replicates in each of three vegetation zones, Spartina alterniflora, S. patens, and a transition zone. Three sites were sampled simultaneously, either within or across vegetation zones, using a dynamic flux chamber technique. Difficulties with calibration and field equipment resulted in fluxes with maximum absolute uncertainties of greater than plus or minus 200 percent. However, the relative uncertainty between subsequent samples was closer to plus or minus 20 percent. Chambers are expected to affect the natural flux of gases by altering the humidity, temperature, and composition of the gas inside the chamber. Summertime fluxes are highest for all gases except COS which demonstrated evidence of a springtime peak. A summertime background flux of 5 to 100 x 10(exp -9) g S/m(sup -2) min was observed for all gases, while S alternifora fluxes of MeSH and DMS were approximately 8 fold and approximately 100 fold higher than S. patens, respectively. DMS and MeSH fluxes were higher during the day than at night. Evidence of COS uptake by plants was observed. CS2 appeared to be the quantitatively least important sulfur gas emitted. Improved laboratory and sample collection techniques and further data collection in the field will yield information on the details of salt marsh variability, will improve estimates of the error associated with single flux measurements, and will allow a more accurate estimation of sulfur fluxes from salt marshes based on vegetation and area coverage data. Inability to control the temperature and humidity inside of the chamber remain significant problems with the chamber design.

Morrison, Michael Cope↗

Factors controlling sulfur gas exchange in Sphagnum-dominated wetlands

Atmosphere-peatland exchange of reduced sulfur gases was determined seasonally in fen in NH, and in an artificially-acidified fen at the Experimental Lakes Area (ELA) in Canada. Dimethyl sulfide (DMS) dominated gas fluxes at rates as high as 400 nmol/m(sup -2)hr(sup -1). DMS fluxes measured using enclosures were much higher than those calculated using a stagnant-film model, suggesting that Sphagnum regulated efflux. Temperature controlled diel and seasonal variability in DMS emissions. Use of differing enclosure techniques indicated that vegetated peatlands consume atmospheric carbonyl sulfide. Sulfate amendments caused DMS and methane thiol concentrations in near-surface pore waters to increase rapidly, but fluxes of these gases to the atmosphere were not affected. However, emission data from sites experiencing large differences in rates of sulfate deposition from the atmosphere suggested that chronic elevated sulfate inputs enhance DMS emissions from northern wetlands.

Demello, William Zamboni↗