This review focusses on the use of recycled and virgin polymers in mineral and metallurgical processing, both high and ambient temperature processes, including novel applications. End of life applications of polymers as well as the utilisation of polymers during its life time in various applications are explored. The discussion includes applications in cleaner coal production, iron and steel production, iron ore palletisation, iron alloy manufacturing, manganese processing, E-wastes processing and carbon sequestration. The underlying principles of these applications are also explained. Advantages and disadvantages of using these polymers in terms of energy and emission reductions, reduction in non-renewables and dematerialisation are discussed. Influence of the polymers on controlling the evolution of micro and nanostructures in alloys and advanced materials is also considered.  Polymers or macromolecules are derivatives of petroleum or natural gas characterized by long chain molecules, classified as elastomers (rubbers), plastics and fibers. They are the second-most important basic materials and are expected to exceed a global production of 420 million tonnes annually in 2020 . The indConcerns are mounting regarding the usage, disposal and accumulation of EOL plastics in landfills and in natural habitats. Ingestion or entanglement in plastic and leaching of chemicals from plastic products can affect wild and marine life and humans. Currently, there is a global consensus that plastics are a threat to the environment, prompting many governments to ban single use plastic products, including Australia, India, Kenya, U.K., Taiwan, Zimbabwe, Montreal (Canada), Malibu and Seattle (USA), Hamburg (Germany), France, Morocco, and Rwanda. ➢Land filling results in no net greenhouse gas (GHG) emissions, no GHG benefits and a waste of valuable resources .➢Mechanical recycling corresponds to the reprocessing of plastic waste to produce new products.➢Energy recovery option is used to generate heat, steam or electricity or as solid refuse fuel for co-fuelling of blast furnaces or cement kilns, or pyrolysis to diesel fuel or gasification. Highly mixed plastics, such as some electronic and electrical wastes and automotive shredder residues, are the candidates.➢Pyrolysis, a recycling technique, includes non-catalytic thermal or catalytic thermal methods. High-temperature pyrolysis of plastics is particularly advantageous for metallurgical processes running at very high temperatures, e.g., the Corex Process, blast furnace, HTK Process  of Voestalpine-Österreichische Mineralölverwaltung (VOEST-ÖMV), and the Thermoselect Process . Unsorte▪Gasification of plastics occurs approximately at 525–625 °C, much before the steel melting temperature . It . It 6]. This review will focus on the specific applications, relevant plastic types, distinct properties of plastics or interactions among them and recycling and energy recovery of the relevant EOL polymers in mineral processing and metallurgy. Energy and fuel: Plastics have a high calorific value when compared with other materials , making them a convenient energy source.4, CO, H2 and the char produced during the pyrolysis of the plastics can replace the coal-based coke as a reducing agent in blast furnace applications.Reductant: The CHBinder: Virgin thermoset polymers and inorganic silicones are adhesives and adhesion promotors (coupling agents). They form strong interfacial chemical bonds with transition metal oxide and impart high strength in the pellets using these thermosets as the binder. While epoxy thermoset resins lend high compression strength, the silicones, on the other hand, are easily compressible; nevertheless, they form stronger bonds with metal oxide and do not crumble. In addition, they also offer other advantages of the plastics as an energy source and as the reductants. They offer energy savings, better mechanical properties compared to the conventional binders, such as bentonite, which due to the high silica content, adds to the slag burden. Thermoset polymers offer high strength to the pellets under ambient conditions and can replace the conventional binders, such as bentonites, and organic binders in palletisation process .8 with nano- and microstructures.Alloying material: Virgin thermoset polymers and inorganic silicones also act as alloying agents in ferrosilicon and silicon carbide, and iron carbide. Silicones can substitute coke and quartz in iron alloy (ferrosilicon) production . The visCarbon sequestration: Virgin thermosets can completely consume the carbon dioxide produced in a high carbon footprint reaction, such as magnesia and magnesium and clinker production, greatly reducing the global warming potential by up to 99%. This is achieved through the use of thermosets for carbon sequestration in the thermal decomposition of magnesite [Application in dewatering of coal: High moisture content in coal, in addition to detrimentally impacting the calorific value, also attracts high transportation tariffs and energy penalties when evaporating techniques are employed for dewatering. Superabsorbent polymers (SAPs) offer ambient dewatering of low-grade coals, greatly improving their calorific values and reducing the transport tariffs. Use of SAPs to dewater fine black coals, brown coals and tailings has been reported ,11,12.Applications of polymeric materials in mineral processing and metallurgy are determined by their pyrolysis products, bonding capacity with metal oxides as the alloying material and the super affinity to the water. In this context, this section discusses the relevant aspects of thermoplastics, thermosets, silicones and super absorbent gels. High-temperature pyrolysis of plastics is particularly advantageous in connection with metallurgical processes running at very high temperatures, e.g., the Corex process, the blast furnace, the recycling of the automotive wastes, the HTK Process  of VOEST-ÖMV, and the Thermoselect process . This paPyrolysis of plastics, mainly the thermoplastics, and to an extent, thermosets are particularly advantageous in connection with metallurgical processes running at very high temperatures. Thermodynamic calculations of the pyrolysis of plastics show only two stable phases, solid carbon and the gas phase, in which two species, namely methane and hydrogen, dominate depending on the temperature; higher-grade hydrocarbons and pyrolysis oil are a possibly thermally unstable third phase . The pyrPyrolysis of thermoplastics yield more volatiles and less carbon residue (less carbonization), whereas, thermosets yield more carbon residue. Thermoplastics also show better wetting characteristics whereas thermosets show little or no wetting characteristics as determined by their bonding characteristics.Thermoplastics  undergo Pyrolysis results in scission and/or cross-link products. Thermoplastics mainly undergo bond or chain scission reactions, involving breaking of the bonds (endothermic) of the long chain backbone, e.g., C–C and C–H bonds depending on the bond energies. Low thermal stability of polyethylene (PE) is attributed to the low bond energies of C–H and C–C bonds, i.e., 406 kJ/mol and 347 kJ/mol, respectively. Thermal processing of thermoplastics such as HDPE and LDPE at 550 °C shows no solid fraction (coke) . The sciPolyalphamethylstyrene undergoes “specific” chain scission with breakage at the chain ends to depolymerize into constituent monomers. Thermal stability of majority of polymers, e.g., PE, PP (polypropylene), PET , PMMA and PC (polycarbonate) reduces in air due to the presence of oxygen as compared to an inert atmosphere. Thermoset plastics, e.g., epoxy-based products and composites, once manufactured, cannot be re-formed or re-used as they are intractable by design . ThermosThe cross-linking reaction is preceded by scission. Cross-linking in most cases is reflected in the strength, toughness and thermal stability of the polymers, which in turn depends on the molecular weight and the length of the polymer chains. High molecular weight and the size of the macromolecules and the intermolecular forces, e.g., hydrogen bonding enhances thermal stability. Cross-linking involves recombination of gases, liquids and solids resulting in non-condensable gas fractions, solid fractions including carbon, and liquid fractions   Table 3. Cross-linked structures that possess high thermal stability require the simultaneous breakdown of several bonds in order to reduce the molecular weight facilitating char formation. Physical characteristics of the chars determine the rate of thermal decomposition of the remainder of the plastics. Char formation as depicted in The yields from the pyrolysis of nonindustrial waste plastics are ≈20% coke, ≈40% tar and light oil, and ≈40% gases . The liq−1 [Epoxy thermoset polymers as adhesives find application in metallurgical processing . Epoxies−1 . Epoxy resins comprise at least two epoxy groups (a three-member ring) per molecule. Epoxy resins contain aliphatic, cycloaliphatic or aromatic backbones. The oxirane or the epoxy ring  can reac4, H2 and carbon residues, and thus can substitute for metallurgical coke and silica in blast furnace operations. The current recycling practice of landfilling or catalytic depolymerisation of silicones [Silicones (organic–inorganic polymers)  are a goilicones ,31 resul−1. The Si–C bond has a bond energy of 318 kJ·mol−1, slightly lower than a C–C bond, while the Si–Si bond is weak (193 kJ·mol−1). An Si–H bond (bond energy of 320 kJ·mol−1) is more reactive than the C–H bond of hydrocarbons (bond energy of 416 kJ·mol−1). The Si–O bonds in silicones are strongly polarised, compared to C–O bonds in organic polymers, with stronger intermolecular interactions [−1 compared to that of siloxane-metal oxide (≈450 kJ·mol−1). The Si–O bond in silicones can replace the metal–oxygen–hydrogen bond or even the carbon–oxygen–metal bond easily through strong surface bonds [−1. Bond strengths at the interface are listed in There is an opportunity to use silicones as coupling agents or adhesion promotors in metallurgical processing. Silicones form strong intermolecular interactions and chemical bonds with inorganic materials, such as glass, silica, alumina, talc, clay, mica, aluminium, iron, titanium oxide, zinc oxide, iron oxide, graphite, carbon black and calcium carbonate, showing improved wettability, compatibility, heat resistance up to 315 °C, weatherability and moisture resistance . Siliconractions . The epoce bonds  resultin2 emission cuts realized in the three regions amount to an estimated 54 million tons per year [Use of virgin silicones provide a reduced carbon footprint for many essential products and services during their life cycle as per the studies conducted in North America, Europe and Japan. Use of virgin silicones, siloxanes and silanes in energy enabling technologies, energy and materials results in energy savings and greenhouse gas (GHG) emissions reductions. The benefit/impact ratio = 9, calculated by dividing the benefits by the impacts from production and end-of-life; when using silicones, siloxanes and silanes outweighs the impacts of production and end-of-life disposal . The benper year . Super absorbent polymers (SAPs)  offer a N-isopropylacrylamide) and cross-linked copolymers of 97% N,N-diethylacrylamide and 3% sodium methacrylate) absorb water and swell at 25 °C and are regenerated by heating to 33 °C and 55 °C, respectively, when they collapse releasing the water.The amount of water that a specific SAP can absorb depends on its chemical composition and morphology as well as the quality of absorbed water , particuThis section discusses in detail the specific applications of the polymers in mineral and metallurgical processing outlined in Dewatering of fine coal, an important aspect of coal cleaning, adds significant cost to the price of clean coal. Victoria has a reserve of 430 billion tons of brown coals, contributing to 92% of electricity generation in Victoria. However, it has a high moisture content that reduces its net calorific value, and thus is a major contributor to total GHG emissions, besides adding to high transportation costs and energy penalties. The high heat capacity of water inhibits use of evaporative drying technologies due to high energy penalties. Capital expenditure (CapEx) of conventional dewatering techniques are: for the centrifuge technique: < Au$50,000/t/hr/rel. reduction in total moisture content (TMC), for vacuum filtering: Au$450,000/t/hr/rel.reduction in TMC, for pressure filter technique: <Au$350,000/t/hr/rel. reduction in TMC, and for briquetting: <Au$350,000/t/hr/rel. reduction in TMC [Inter-particle capillary and/or pendular moisture, which can be removed by mechanical techniques broadly classified as centrifugal, vacuum or pressure dewatering processes.Surface and intra-particle moisture , which can be removed by thermal drying.Chemical water, which is held in chemical combination with certain mineral matter in the coal and may only be released during combustion or gasification process.Types of moisture distribution within fine coals are :Inter-paDzinomwa, et al. (1997)  employedVictorian brown coals have high oxygen and moisture contents and hence low calorific value (≈8000 kJ/kg) before drying or dewatering. However, on drying or dewatering, the calorific value is high . Osmotic dewatering of brown coals using anionic SAP carried out under ambient conditions has no associated energy penalties. Approximately 57% of water is removed within four hours of SAP contact under ambient conditions ,45. This➢Swelling properties (attributed to presence of hydrophilic groups in the network).➢Cross-linking density .➢Structural integrity .➢The “availability” of target water, i.e., how tightly it is bound to the drying substrate.Mechanism of water removal is as follows: dry SAP having higher concentrations of “free” COO– groups than the coal removes moisture preferentially from coal through osmosis . FactorsCoke is utilised as an iron ore-reducing agent in blast furnaces, hydrocarbon oil as raw materials for the chemical industry and the gas as a fuel for power plants. Caking coals, e.g., many bituminous coals, when heated, exhibit thermoplastic effects, i.e., they soften and form a plastic mass that swells and resolidifies into a porous solid. Strongly caking coals, which yield a solid product (coke) with properties suitable for use in a blast furnace, are called coking coals. Japan uses waste plastics to produce coke, coke oven gas (COG) and hydrocarbon oil in a coke oven ,49. NippThe effect of plastic addition on the coal-caking property varies with the type of plastics . The add2 emissions by coke oven chemical feedstock recycling depends on the calorific values and the coke product yields of each plastic resin. For example, in the case of coke oven chemical feedstock recycling, the reduction potential of PS and PP is larger than that of PE [Coke is produced in a coke oven by carbonizing, i.e., heat treating the coal without oxygen at a high temperature. Coke can be produced via the chemical feedstock recycling of waste plastics in the coke oven . When agat of PE .Energy recovery from waste plastics and feedstock recycling are the underlying principles of the coke oven process.Use of both recycled and the virgin polymers in iron and steel making is discussed in this section.2 emitters. Globally, between 4 and 7% of the anthropogenic CO2 emissions originate from this industry [Annual production of steel is about 1.3 billion tonnes worldwide . The iroindustry . Iron ore is reduced with coke in a blast furnace, and liquid steel in a basic oxygen furnace (BOF) is produced from the reduced hot metal (pig iron) mixed with 30% steel scrap. In the electric arc furnace (EAF) process, the iron input is typically in the form of scrap, direct reduced iron (DRI) and cast iron. To produce 1 ton of hot metal, approximately 370 kg of coke plus 90 kg of heavy oil are used depending on the quality of the iron ore and the process used. The 90 kg of heavy oil can be partly substituted with up to 70 kg of plastics waste ,54.The high temperature processability of the thermoplastics  finds ap2 content within and in the off gas exiting the blast furnace. The increased bosh gas H2 reduces the pressure drop to allow a greater gas flow for the same pressure by decreasing the bosh gas density. Injecting waste plastics lowers CO2 emissions by about 30% in comparison to coke and coal [Plastics contain nearly 3 times more hydrogen than the pulverized coal and use of plastics increases the Hand coal . 2 emission reduction potential in blast furnace feedstock recycling depends on the calorific value and the carbon and hydrogen content of the individual plastic resins. Thus, PE has the largest CO2 emissions reduction potential. The CO2 emissions reduction potential of PP is less than those of PE and PS, while PET has negative CO2 emissions reduction potential. This leads to a difference in the coke substitution effect by each plastic resin [4 from waste plastics on the liquid iron surface (Reaction (3)). The presence of H2 was found to aid the carburization, probably through the removal of adsorbed oxygen from the metal surfaces [The COic resin . The meaic resin . The ratsurfaces . Carbon surfaces .CH4 = CIn EAF steel making, replacing ≈30 % of the coke and coal by polyethylene wastes resulted in energy savings up to ≈12 kWh/t of plastic charge . Tyre in2/g in two minutes of the reaction [While extensive use of thermoplastics as a partial substitute for coke in iron and steel industries has been reported, the use of thermoset plastics has not been reported to the same extent. Dhunna et al. (2014)  studied reaction . 4 and H2) from the Bakelite thermosets contribute to the reduction efficiency. High temperature char formation from Bakelite helps increase the carbon dissolution in the liquid iron phase [4 and H2, in addition to being a high temperature  process. It is not clear why the CH4 and H2 evolved from the Bakelite does not help carbon dissolution nor why only Bakelite char contributes to the carbon dissolution and not raw Bakelite (Reaction (4)).The above study suggests thermoplastics perform better in terms of reduction and carbon dissolution compared to the thermosets. The ability of the thermoplastics to melt or undergo phase transformation from solid to the rubbery state above the glass transition temperatures offers a different mechanism compared to the thermosets, which cannot undergo phase transformation. A solid–liquid–gas system provided by thermoplastics promote intimate contact and wetting between the reactants, which the thermosets are incapable of. However, the volatiles  due to their inability to reform to provide the required intimate contact or wetting. Use of virgin thermoset polymers, on the other hand, serves as a binder, reductant, alloying material and fuel, thereby, minimising the need for non-renewable resources such as coke as the reductant and fuel. Iron ore palletisation using epoxy as the binder instead of conventional bentonite or organic binders shows many desirable features . Thermoset polymers like epoxies are low molecular weight viscous polymers. During chemical reactions with curing agents , they undergo further polymerisation and phase transformation from a viscous state to a dry, solid three-dimensional cross-linked molecular network. The iron ore pellets produced under ambient conditions using the thermoset epoxy resin showed a high compression stress of 4 kN without any heat treatment compared to the industry requirement (2.5 kN) of an indurated pellet processed at 1200–1350 °C. The pellets are reported to be non-abrasive, non-sticky and devoid of undesirable fines. The process spells cost, energy and emission reductions for iron and steel industries . The stuEnergy consumption during heat induration of magnetite concentrates ranges from 320,000 kJ/t to 740,000 kJ/t pellets, and it is higher for higher non-magnetite ores due to the absence of exothermic and oxidation reactions. Cold-bonded pellets using epoxy binders, being a low temperature alternative to heat-indurated pellets, consume less energy for bonding self-reducing pellets and for refractory ores that are difficult to heat treat. The estimated cold-bonding capital and operating costs is 2/3 that of heat induration. 2O3 to C and a binder. However, in a recent study, the binder acts as the reductant and fuel at ≈1:0.3 Fe2O3 to C, i.e., at least 10 times less carbonaceous material [2 and CO from the methane generated at the reaction temperatures, which aid the reduction reaction, as well as the solid carbon residue [Use of virgin epoxy resin promotes dematerialization in terms of carbonaceous materials and the binder. The conventional iron ore reduction/palletization requires at least 1:3 Fematerial . The pyr residue . The higThe mechanism involved in producing pellets of high strength can be explained as follows: Viscous adhesive (epoxy resin) is capable of dissolving and diffusing into iron oxide ,72. The 2) with carbon in the presence of iron ore, scrap iron, mill scale or other source of iron at temperatures greater than 1800 °C.Silicon carbide (SiC) and ferrosilicon (FeSi) find application in the clean energy systems such as photovoltaic industries, and economical production of high purity hydrogen. It is estimated that the global demand for silicones will rise 6% per year to 2.4 MMT (million metric tonnes) in 2018. SiC is produced from petroleum coke and a silica or quartz sand at high temperatures (1700–2500 °C). FeSi is produced industrially via the carbo-thermic reduction of silicon dioxide (SiOSilicones are used in the production of ferrosilicon and the epoxy resin (Epiglue) having silica as the filler are used for producing the silicon carbide . The stuA carbothermal reduction process requires intimate contact between a solid oxide and a carbonaceous material  facilita2 is less than the stoichiometric requirement of 3, Si is lost as SiO. While it is expected the carbon deficit could be compensated by introducing methane gas to increase the SiC content, it was observed [Presence of methane is reported to increase the SiC content, when supplied externally together with hydrogen. However, when C/SiOobserved  that the4, H2, C and Si, in addition to facilitating the reduction of the iron oxide, aid the dissolution of carbon and silicon in the iron matrix to form SiC, FeSi and FeC8. Important technical aspects in this context are [SiC and FeSi formation does not require any high-purity quartz or coke.Solid-liquid-gas reactions facilitated by the viscous polymer resin help achieve high reaction efficiency.2, CH4, C and Si produced in situ are derived from virgin polymers resins.Resources such as HThere is a potential to recover these resources from feed stock recycling of thermosets and inorganic–organic polymers like silicones. However, it is necessary to make sure intimate contacts between the reactants are facilitated.Iron oxide–polymer composites as potential replacement for coke as spacer.Nano- and microstructured alloy phases are formed.However, a solid-liquid-gas reaction facilitated by the viscous virgin polymer resin with the reactants can ensure intimate contact between the SiO and the insitu production of methane can increase the SiC content . The pyrThe reaction mechanism of alloy formation can be explained as follows: the virgin epoxies and the silicones in the fluid or viscous form prior to curing facilitate solid–liquid–gas system providing intimate contact between the reactants. They thoroughly wet the reactants and form interfacial covalent bond between the reactants upon curing a,b 29,8,829,83. 2 to Si from the slag [2 emission as well as the evolution of CO and CH4 indicated the thermal decomposition of the polymer to liberate significant amounts of CH4 [2 using mechanically recycled PP utilisation in the pre-reduction step (MnO2 → Mn2O3/Mn3O4 → MnO) is demonstrated in Reference [4 as reductants [The reduction of MnO in slag by blends of coke and high-density polyethylene (HDPE) shows improved wettability and reduction efficiency compared to reduction by pure coke. Increasing HDPE content resulted in further improvement in reduction and wettability and allowed for the near complete reduction of MnO and partial reduction of SiOthe slag . Decreass of CH4 . The proeference . Howeverductants . Reductiductants .Mechanical recycling of PP and the feedstock recycling of HDPE and the PP are employed in the manganese processing.It is predicted that 49.8 million metric tons of e-waste will be produced globally in 2018 and 52.2 million metric tons by 2021 . PotentiE-waste is a source for toxic compounds such as antimony, lead, mercury, cadmium and brominated flame retardants; reclaimable materials include a mixture of metals such as copper, aluminium, steel, gold, silver and platinum group metals and various types of plastics and ceramics. The material composition of the electronics is given in The integrated furnace has necessary installations to collect the off-gases.The plastic fraction serves as a replacement of coke either partially or fully as a reducing agent as well as the fuel.The rapid heating above 1000 °C will contain the emissions of Dioxin/Furans within the acceptable limits.The copper/nickel/lead base metal metallurgy allows the precious and the lead special metals to be collected as slag.The smelter separates precious metals in a copper bullion. Other metals concentrate in a lead slag are further processed at base metals operations.The ceramics and the glass are recovered as a slag.E-wastes are processed in large-scale integrated energy- and resource-efficient smelters/plants operated by multinational companies, such as Boliden Minerals (Sweden), Norddeutsche Affinerie (Germany), Union Minière (Belgium), Noranda (Canada) and Outokumpu (Finland). They use a combination of different recovery methods such as mechanical recycling, feedstock recycling and energy recovery. Integrated processing of e-wastes increases eco-efficiency, which otherwise could be lost in the isolated steps. Main features of integrated processing of e-wastes are:At Noranda reactor recycling facility, about 100,000 tons of e-waste is processed for metal recovery. Energy usage in the Noranda reactor is reduced due to the combustion of plastics in the e-waste feedstock . UMICOREThe Kaldo furnace can recover metals from more than 100,000 tons of secondary raw materials from cable scrap and printed circuit boards. Plastics are used as fuel and easily oxidisable impurities are dissolved in a liquid slag and precious and base metals are collected as an alloy or “Matte” (liquid sulphides) . The rolBoliden’s Rönnskär smelter, in northern Sweden  has been recycling various waste materials since the 1960s. The smelter’s annual capacity for recycling electrical material is 120,000 tonnes, sourced primarily from Europe. Plastic in the e-waste acts as a source of energy during smelting and generates steam that is converted into electricity or district heating. Boliden has the technical capability to take the entire shredded PC scrap into their zinc fuming plant without extensive dismantling. The zinc fuming furnace can effectively reduce the dioxins and furan content when processing the E-wastes . The higThe zinc fuming plant consists of three separate processes, namely, the fuming furnace, settling furnace and the clinker furnace. The reduction process in the fuming furnace reduces metal oxides such as magnetite, lead oxide, zinc oxide, lead and arsenic, and extracts halogens from the slag. The resulting mixture of oxides is de-halogenated in the clinker furnace. The subsequent process of halogen removal in the clinker furnace can handle increased load of halogens from the steel making dust and other feed streams. Halogen removal is carried out in the clinker furnace by re-heating the raw fume together with coke additions to approximately 1200 °C. The zinc thus produced show dioxins values below the detection limits. The total halogen content liberated in the fuming plant is neutralised in the gas cleaning section . The totThe heat balance analysis of plastic substitution from e-waste in comparison with normal operations using coke suggests plastics are used as the chemical feed stock, where carbon and hydrogen from the plastics substituted for the carbon and hydrogen provided by the coal for reducing the slag. This conclusion is arrived at by comparing the fuming speed, the generation of steam, and the specific energy as shown in Mechanisms of e-waste recycling involves combination of recovery methods such as mechanical recycling, feedstock recycling, thermal depolymerization and energy recovery. Plastic components of e-waste cannot be easily recycled due to the mix of flame retardants, pigments and mixed types of plastics. The most common outlets for these mixed fractions are landfill and incineration plants, which are becoming increasingly restricted and costly given recent legislative developments. Where re-use of plastic components from the e-wastes is not possible, mechanical recycling is opted. In mechanical shredding and sorting processes for e-scrap (pre-treatment), large volumes of various mixed plastic fractions are usually generated. Often, it is not economically viable to separate these further and recover the various types of plastic. There is also a difficulty of achieving acceptable product quality. Limited markets for recycled polymers and innovations in polymer performance and consumer acceptance add to the problems. This is because of the contamination during sorting process of plastics and subsequently during the recycling process affect the product quality adversely. Furthermore, all residual metals still contained in them would inevitably be lost. Separating residual metals by mechanical means can become quite costly while generating only little additional metal value. Mechanical recycling of older WEEE which contains plastics cause problems when dismantling processes do not utilize adequate means, facilities and trained people. 2, or as fuel [Plastic content in the e-waste is used either as a chemical feedstock to replace the reducing agents, CO and H as fuel  due to t as fuel , which c2 emission. The main environmental concerns to produce different types of MgO are the use of energy and emissions to air. The calcination process of magnesite has a high carbon footprint. It is the principal user of energy and the main source of emissions, with 1.1 t CO2 resulting from the carbonate decomposition to produce 1 t of magnesia (MgO) accounting for direct CO2 emissions of 6.7 Mt from the calcination process alone. A study reports use of virgin epoxy resin and biomass can reduce 99% of the carbon dioxide produced in thermal processing of magnesium carbonate while simultaneously producing hydrogen [Global growth in the magnesium market is expected to reach 1.2 Mt/year by 2020 . Application of Mg has been limited by relatively high cost of production, associated energy costs and a high CO3, dead burnt MgO production and magnesia carbon refractory bricks production in presence of plastics and biomass with synergistic effects. The current operating temperature of ≈1600 °C can be brought down by at least 100–200 °C due to the reducing gases produced by the plastics and the biomass enhancing the energy efficiency of the process [3:biomass:epoxy resulted in H2 enriched product paving way to cleaner hydrogen economy.Magnesium metal production (where the intermediate product can be magnesia as described in the research) is another high carbon footprint reaction. Use of plastics can greatly reduce the emissions, energy and process temperatures not only during the intermediate MgO formation, but also during the carbothermic reduction to magnesium metal, eliminating the need for the non-renewable coke. The above study  suggests process . Magnesi2 emissions. Two thirds of this (0.94 Gt per year in 2005) is generated by the decomposition of limestone into cement clinker and CO2. The remaining one third is from fuel combustion. The total CO2 emissions during the cement making process are mainly due to:raw material decomposition (59%),fuel combustion (26%), andelectricity consumption (12%).Cement production accounts for about 22% (1.5 Gt in 2005) of the industry sector’s total direct CO2 emission factor is 0.81 t/tonne cement. Producing one tonne of clinker will have: 1 t × 65% × 0.79 = 0.51 t of CO2 emitted from CaCO3 decomposition. The magnesium and calcium have similar chemistry and a similar reduction in emissions, global warming potentials, energy and process temperature can be achieved in Clinker production. Use of plastics and biomass can reduce the emissions during raw material decomposition in addition to energy recovery.The overall CO4, H2, CO, CO2 and other hydrocarbons used as resources in high temperature metallurgical processing, e.g., in blast furnace and EAFs. In Japan, mechanical recycle, feedstock recycle, and energy recovery were used to increase the waste plastic utilization over 50 wt. % ratio to solve the problem of shortages in landfills and incineration sites. Consumable carbon anodes for the aluminium industry using high temperature interactions of waste plastics with petroleum coke have been developed [Plastics, used or virgin, have a higher calorific value than coal and can provide a much-needed local energy supply . Pyrolyseveloped .The high temperature processability of the thermoplastics finds applications blast furnaces, especially iron and steel processing ,56. Wast2 emissions by about 30% in comparison to coke and coal depending on the type of polymers used as shown in At the outset, injecting waste plastics, which have a higher calorific values (CV) than coal, decreases blast furnace coke and energy consumption and lowers COReaction (5) occurs at temperatures > 700 °C; Reaction 6 occurs at temperatures < 580 °C ,102.2 emission reduction potentials, i.e., there is an increase of CO2 emission  compared to MnO reduction by methane (928 °C), as shown in Reactions (7) and (8) ,107:MnO −1 [The Gibbs free energy change, ΔG° for the above reaction is 257.75−0.1598T J·mol−1 .MnO +10−1.ΔG° = 377.68−0.3144T Jmol4-H2 gas relative to graphite increases with temperature. This makes the reduction of manganese ore by methane-containing gas faster than in the conventional carbothermal reduction process [The exponential increase in the increasing values of equilibrium constants for Reaction (8) is 8.46 at 1000 °C, 113.80 at 1100 °C and 1075 at 1200 °C . The inc process . The pellets using epoxy resin as the binder show high compression strength of 4 kN without heat treatment compared to the industry requirement (2.5 kN) of an indurated pellet processed at 1200–1350 °C. Energy consumption during heat induration of magnetite concentrates ranges from 320,000 kJ/t to 740,000 kJ/t pellets and higher for higher non-magnetite ores due to the absence of exothermic and oxidation reactions. Cold bonded pellets being a low temperature alternative to heat-indurated pellets consume less energy for bonding self-reducing pellets and for refractory ores that are difficult to heat treat. The estimated cold bonding capital and operating costs is 2/3 of that of heat induration [The FeSi and SiC formation would normally occur at >1800 °C in conventional process. Use of virgin silicones and epoxy resins has seen FeSi and SiC formations around 1500 °C . In EAF Chlorinated polymers like PVC may lead to dioxin formation during landfilling and incineration. Exposure to dioxins can cause skin lesions, damage the immune system, interfere with hormones and also can cause cancer. However, dioxins are contained within the required level during pyrolysis of waste plastics. Heavy metals emissions, especially the Cd and Hg from the blast furnaces, could pose problems when the plastic wastes are sourced from mechanically separated packaging and commercial wastes and the lighter fractions from the end of life vehicles . HoweverCoal dewatering, where super absorbent polymers offer ambient non-evaporative technology to dry low-grade brown coals and fine black coals saving on costs, energy and emissions while simultaneously enhancing the calorific values of the same. These super absorbent polymers can be regenerated at low pH or low temperatures (37–52 °C)2 emissions >30%. In EAF, they aid slag foaming contributing to energy reductions. Harmful emissions of dioxins are reduced during the feedstock recycling of waste plastics.Chemical feedstock recycling of waste plastics in coke oven and blast furnaces produce carbon residues and reducing gases such as methane and hydrogen and fuel oils. They reduce the coke consumption in blast furnace iron making and COFor iron ore palletisation in this novel process, the epoxy resins used as the binder, reductant and as fuel, offer high compression strength under ambient conditions, far exceeding the heat induration strength, saving on costs, energy and emissions. In addition, the reducing atmosphere provided by the polymer resins reduce the reaction temperature by about 100 °C. Use of the bio-renewable epoxy resins, currently being commercialised, makes this a green process.Iron alloy production, where silicones and the epoxy resins are used as the alloying agents, binder, reductant and fuel towards the production of ferrosilicon, silicon carbide with varying microstructures. The alloy formation occurs at 1600 °C as opposed to the conventional 2000 °C.The polymer resins reduce the non-renewable coke consumption in the above processes.2 produced in a high carbon high footprint reaction, such as thermal decomposition of magnesite or clinker production, simultaneously producing hydrogen, contributing to hydrogen economy.Carbon sequestration, where the plastics used totally consume the COThe plastics in e-wastes are used as fuel and reductant in recovering valuable metals.Recycling of used polymers offers competent waste management strategy reducing the land-fill burden.Concerns are mounting regarding the usage, disposal and accumulation of EOL plastics in landfills and in natural habitats. Ingestion or entanglement in plastic and leaching of chemicals from plastic products can affect wild and marine life and humans. Currently there is a global consensus that plastics are a threat to the environment prompting many governments to ban single use plastic products, including Australia, India, Kenya, U.K., Taiwan, Zimbabwe, Montreal (Canada), Malibu and Seattle (USA), Hamburg (Germany), France, Morocco, and Rwanda. About 4% of world oil production is used as a feedstock to make plastics and a similar amount is used as energy in the process. When one third of the plastics produced are used in packaging and other short-lived applications only to be rapidly discarded , such us


Ionic liquids (ILs) as designer solvents have been applied in biomass pretreatment to increase cellulose accessibility and therefore improve the enzymatic hydrolysis. We investigated the characterization of the micromorphology and the topochemistry of poplar wood during 1-ethyl-3-methylimidazolium acetate pretreatment with mild conditions (90 °C for 20 and 40 min) by multiple microscopic techniques . Chemical composition analysis, XRD, cellulase adsorption isotherm, and enzymatic hydrolysis were also performed to monitor the variation of substrate properties. Our results indicated that the biomass conversion was greatly enhanced (from 20.57% to 73.64%) due to the cell wall deconstruction and lignin dissolution (29.83% lignin was removed after incubation for 40 min), rather than the decrystallization or crystallinity transformation of substrates. The mild ILs pretreatment, with less energy input, can not only enhance enzymatic hydrolysis, but also provide a potential approach as the first step in improving the sequential pretreatment effectiveness in integrated methods. This study provides new insights on understanding the ILs pretreatment with low temperature and short duration, which is critical for developing individual and/or combined pretreatment technologies with reduced energy consumption. Biofuels are considered to be the main potential replacement for fossil fuels in the near future. Liquid biofuels like bioethanol can be produced via the fermentation of sugars produced from the cellulose and hemicelluloses within the lignocellulosic biomass, such as wood and agricultural or forest residues. These abundant, readily available, and low-cost lignocellulosic materials can capture huge amounts of solar energy and therefore play a crucial role in the development of second-generation bioethanol. However, unlike the starch from corn or other grains that is susceptible to hydrolysis, it is hard to hydrolyze the lignocellulosic biomass enzymatically due to ‘biomass recalcitrance’, the natural resistance of plant cell walls to microbial and enzymatic deconstruction . TherefoA number of pretreatment technologies have been developed in the past years and reviewed by researchers ,3,4,5,6.Ionic liquids (ILs) are organic salts that usually melt below 100 °C; they consist of a large organic cation and a small organic or inorganic counterion. Due to their high thermal stability, nearly complete non-volatility, wide liquid range, and excellent solvation properties, ILs are becoming attractive alternatives to volatile and unstable organic solvent . MoreoveIn the previous studies, most of the ILs pretreatment of biomass or cellulose prior to enzymatic hydrolysis or second-step pretreatment in combined methods is performed at a temperature higher than 100 °C and with a long duration ,18,19. LThe objective of this study is to reveal the mechanism of mild [Emim]Ac pretreatment for improving the enzymatic hydrolysis of biomass with less energy input by combing the information about the microstructure and the topochemistry of pretreated poplar wood. Multiple microscopic techniques, chemical composition analysis, powder X-ray diffraction (XRD), cellulase adsorption isotherm, and enzymatic hydrolysis were performed to monitor the variation of substrate properties. We present evidence that cell wall deconstruction and lignin dissolution during mild [Emim]Ac pretreatment play an important role in improving enzymatic hydrolysis. In addition, this mild IL pretreatment with reduced energy intake may improve the sequential (second-step) pretreatment efficacy in integrated pretreatment technologies, which will be further investigated in future work.The chemical composition content of biomass samples is a significant factor that affects the sequential enzymatic hydrolysis. The chemical compositions of poplar samples untreated and pretreated with IL are illustrated in As mentioned, ILs can dissolve a large number of biomacromolecules such as cellulose, hemicelluloses, and lignin with high efficiency. These chemicals can be regenerated from solutions by adding anti-solvents such as water and ethanol. [Emim]Ac, one efficient solvent in dissolving wood flour, can remove lignin with relatively good selectivity . Table 1An increase of the relative cellulose content of pretreated poplar (from 45.08% to 50.91%) was benefited by the extensive removal of lignin. In fact, little carbohydrates were extracted from the wood flour by [Emim]Ac; the content of glucan, xylan and other sugars changed slightly during [Emim]Ac pretreatment with mild conditions.In order to evaluate the effectiveness of IL pretreatment on cellulose digestibility, the raw poplar samples and the residues after [Emim]Ac pretreatment were subjected to a 72-h enzymatic hydrolysis test. The temporal profiles of cellulose conversion were presented in Lignin reduces the effectiveness of enzymatic hydrolysis by limiting the cellulose accessibility as well as by binding cellulose unproductively. Although the relative contribution of these two roles of lignin is not yet fully understood, the removal of lignin by IL pretreatment in this study has actually improved the enzymatic hydrolysis of poplar samples.2) in the range of 0.987–0.995. The high R2 suggested that the model was adequate for describing the adsorption of cellulase in the present study. As shown in max = 71.15, 76.15 mg/g) than the untreated substrate (Emax = 62.83 mg/g). The Langmuir constant (Kd) and distribution coefficient (Kr) can be used to estimate the affinity of enzymes for substrates Ac pretreatment, field emission scanning electron microscopy (FE-SEM) and bright field microscopy imaging were performed on poplar flour and cross sections. The obtained FE-SEM and bright field microscopy images are shown in FE-SEM imaging technology revealed significant gross morphological changes in the IL pretreated poplar samples. Compared with the densified smooth surface of raw wood particles a,d, the Chemical composition distribution in wood cell walls is another significant factor affecting the cellulose accessibility. CLSM and CRM technologies were often used to visualize the components’ distribution in cell walls before and after IL pretreatment ,21. In tAs can be seen from CRM is another technology used to determine the topochemical changes of plant cell walls during pretreatment . In this−1, which includes bands from the wood components such as cellulose, hemicelluloses, and lignin, is of interest. There is little difference between the Raman spectral features of cellulose and hemicelluloses; the hemicelluloses bands are broader and can reside beneath the cellulose bands as a result of their low amount and amorphous character Ac pretreatment. However, the carbohydrate concentration changed slightly for all the cell wall regions.haracter . Therefoectively ,36. As s−1 and the sloping baseline method. As we can see, the image intensity decreased significantly for IL pretreated samples, which indicates the removal of lignin. Consistent with the results of average Raman spectra, although more lignin was removed from the CCML region, the decline of lignin concentration in the SW region was also remarkable. The band region of 2867–2907 cm−1 was selected to investigate the carbohydrate distribution, combing with the sloping baseline method. As shown in To obtain more information about chemical variation in the cell walls over a large area during IL pretreatment and to enhance visualization of composition  distribution, we used the Raman mapping technique. Chemical images of poplar cell walls obtained by CRM are presented in The delignification of poplar wood greatly increased the yield of enzymatic hydrolysis, because lignin reduces enzyme effectiveness by limiting the cellulose accessibility as well as by binding cellulase unproductively . Howeverv/v) in a Soxhlet apparatus for 6 h. Then, the extractive-free sample was dried for 16 h in an oven at 60 °C for further use. For microscopic measurements, 10 μm-thick cross sections were cut from the secondary xylem of the dry poplar stems by a sliding microtome. The IL 1-ethyl-3-methylimidazolium acetate ([Emim]Ac) was purchased from the Lanzhou Institute of Chemical Physics, Lanzhou, China. The commercial cellulase used in this study was purchased from Novozyme, Bagsvaerd, Denmark, and the filter paper activity of the cellulase was 145 FPU/g.The dry poplar stems about 6 years of age used in this work were collected from the botanical garden of Beijing Forestry University, China. The raw material was knife-milled, and the wood flour (60–80 mesh) was extracted with toluene–ethanol  under stirring. Then, the suspended wood flour was recovered by filtration and washed with deionized water and dried in an oven at 80 °C for 2 h. The mass of recovered wood flour was then determined. Poplar wood sections were placed in 5 mL vials together with 1 mL of [Emim]Ac and were heated in an oven at 90 °C. After incubation for 20 and 40 min, the wood sections were rinsed with deionized water for 10–15 times to replace the residual IL from the samples.The chemical compositions of raw and pretreated poplar samples were determined according to the analytical procedures established by the National Renewable Energy Laboratory (NREL) . The resw/v). The cellulase was employed at the activity of 20 FPU/g substrate for all samples. The reaction mixture was digested in an air-shaking incubator at 48 °C and 150 rpm for 72 h. The IL pretreated poplar samples were rinsed with deionized water 10–15 times before enzymatic hydrolysis. To monitor hydrolysis kinetics, 100 µL of hydrolyzed slurry was taken periodically  and analyzed by HPAEC. Results are presented as the percentage of the corresponding theoretical glucose yield of each sample. All assays were performed in triplicate.Enzymatic hydrolysis of the raw and pretreated poplar samples was carried out in a 25 mL digestion solution containing 0.05 M sodium acetate buffer (pH 4.8), using a 50 mL Erlenmeyer flask at a biomass loading of 2% (w/v) in 50 mM acetate buffer (pH 4.8) at 4 °C for 90 min to reach equilibrium. After incubation, the supernatants were collected and centrifuged at 5000× g for 10 min. The free protein content of the supernatants was measured by Bradford protein assay . The amount of cellulase bound to the substrate was indirectly calculated by subtracting the measured free protein from the total protein added. The experimental data were fit to the following Langmuir adsorption isotherm using software package OriginPro 8.0 :aE is the concentration of bound cellulase (mg/g substrate), Emax is the maximum content of absorbed cellulase (mg/g substrate), fE is the free protein concentration in the supernatant (mg/mL), and dK is the binding constant (mL/mg cellulase). Additionally, the distribution coefficient r(K) was calculated as follows:To measure the cellulase adsorption isotherm, a range of cellulase concentrations (0.01–1.0 mg/mL) were incubated with the untreated and IL pretreated substrates, with a final dry solid content of 2% , employing Ni-filtered Cu Kα radiation (wavelength = 0.154 nm) at 40 kV and 30 mA. Scans were obtained from 5° to 40° 2θ (Bragg angle) at 0.03° per second of scanning rate and at room temperature. Sample crystallinity, as expressed by CrI was measured from the XRD data and calculated with the following equation :(3)CrI=The surface morphological features of raw and pretreated poplar samples were studied by an FE-SEM apparatus  at an accelerating voltage of 5 to 10 kV. The wood flour particles and wood sections before and after IL pretreatment were prepared by vacuum drying. Subsequently, they were mounted on carbon tape-adhered aluminum stubs and sputter coated with 10–12 nm gold particles by using a vacuum sputter prior to acquiring images.The surface of raw and pretreated wood sections was observed by a confocal laser scanning microscope . An oil immersion objective lens  equipped with an MPlan 100× oil immersion microscope objective  was utilized in this study. A linear polarized laser , focused with a diffraction-limited spot size (0.61 λ/NA), was used to conduct measurements. The laser power on the sample was approximately 8 mW. The Raman light was detected by an air-cooled, front-illuminated, spectroscopic charge-coupled device (CCD) behind a grating (1200 grooves·mm−1) spectrometer. For mapping, an integration time of 2 s was chosen and every pixel corresponds to one scan with a spectrum acquired every 0.5 µm by averaging 2 s cycles. Labspec 5 software  was used for spectra analysis and image processing.The raw and pretreated wood sections were placed on a glass slide with a drop of DIn summary, we have illustrated the cell wall deconstruction and lignin dissolution in poplar wood during [Emim]Ac pretreatment with short duration and low temperature, using chemical composition analysis, XRD, cellulase adsorption isotherm, and multiple microscopic techniques . In these mild conditions, although decrystallization or crystallinity transformations were not observed, the enzymatic hydrolysis for the regenerated substrates was greatly improved compared to untreated samples, due to the increased cellulose accessibility and the decreased lignin-binding cellulase as a result of lignin removal. Additionally, the increase of porosity and chemical component dissolution may improve the effectiveness of second-step pretreatment in the integrated methods. The mild ILs pretreatment investigated in this study can not only enhance enzymatic hydrolysis with less energy input, but also provide a potential approach as the first step in improving the sequential pretreatment effectiveness.


GLRA1 accounts for about 80% of cases. Dominant and recessive mutations have been identified in GLRA1 gene as pathogenic variants in many individuals with the familial form of Hyperekplexia and occasionally in simplex cases.Hyperekplexia also known as Startle disease is a rare neuromotor hereditary disorder characterized by exaggerated startle responses to unexpected auditory, tactile, and visual stimuli and generalized muscle stiffness, which both gradually subside during the first months of life. Although the diagnosis of Hyperekplexia is based on clinical findings, pathogenic variants in five genes have been reported to cause Hyperekplexia, of which GLRA1 gene revealed, in both probands, compound heterozygous mutations: c.895C > T or p.R299X inherited from the mother and c.587C > A or p.D98E inherited from the father.In the present study, we describe clinical and genetic features of two Italian siblings, one with the major and one with the minor form of the disease. DNA samples from the probands and their parents were performed by NGS approach and validated by Sanger sequencing. The analysis of the GLRA1 have not been reported before as compound mutations. What clearly emerges within our study is the clinical heterogeneity in the same family. In fact, even though in the same pedigree, the affected mother showed only mild startle responses to unexpected noise stimuli, which might be explained by variable expressivity, while the father, showed no clear signs of symptomatology, which might be explained by non-penetrance. Finally, the two brothers have different form of the disease, even if the compound heterozygous mutations in GLRA1 are the same, showing that the same mutation in GLRA1 could have different phenotypic expressions and suggesting an underling mechanism of variable expressivity.Until now, these two identified mutations in  GLRA1, OMIM 138491) mapping to chromosome 5p33.35 were first reported in 1993 to cause autosomal dominant familial hyperekplexia [GLRA1 mutations have been reported as dominant missense (23%), recessive missense (39%) and recessive nonsense (38%) [SLC6A5 , GLRB , GPHN (gephyrin), and ARHGEF9 (Rho guanine nucleotide exchange factor 9) gene [GLRA1 gene but discordant phenotypes.Hyperekplexia (HPX) also known as Startle disease OMIM 149400) is a rare neuromotor hereditary disorder characterized by exaggerated startle responses to unexpected auditory, tactile, and visual stimuli and generalized muscle stiffness, which both gradually subside during the first months of life 49400 is . Exaggerekplexia . This α12 saturation monitoring during sleep and clonazepam treatment were started, with complete resolution of the episodes. The child developed speech delay in early childhood, and mild behavior disorders  and no motor impairment. We followed-up the patient until he was 17, no more episodes of hypertonia occurred but he reported occasionally startle episodes triggered by sudden acoustic stimulation, vertigo and trembling hands. An improvement in verbal skills was observed after speech therapy. At the age of 17 his neurological exam, social activity and school abilities are normal. CZP treatment is still ongoing. In consideration of the clinical diagnosis in the younger brother and because of the reported episodes, the older proband was examined at our Center, when he was 4 year old. He was born at term, from uneventful pregnancy and had normal neuromotor and cognitive development. Since the age of 2 years, he had been presenting several episodes of limbs hypertonia, associated with loud crying, during sleep and triggered by sudden noise. These episodes slowly decreased over the years, but the child showed episodes of sudden falling and generalized hypertonia that affected his motor performances and social activities. CZP treatment was started with significant clinical improvement. At the age of 10 an attempt to reduce drug dose caused a relapse of his usual startle episodes trigged by sudden noise. All the EEG performed in both patients were normal, except for high frequency rhythm on the anterior regions, due to benzodiazepine treatment. The mother reported mild startle triggered by sudden noise, without impairment in daily activities, so no therapy was prescribed. The father was completely asymptomatic and in good general condition.The pedigree for the family is presented in the Fig. GLRA1, GLRB, SLCA, GPHN, and ARHGEF9) associated with neurological disorders. Genes were sequenced on an Ion Torrent™ Personal Genome Machine™ (PGM) sequencer (ThermoFisher Scientific) with a PCR amplicon-based library preparation (AmpliSeq™) using the ProFlex PCR System (ThermoFisher Scientific) and the Ion AmpliSeq™ Library Kit 2.0 reagents. The amplified library was quantified with the Invitrogen™ Qubit™ Fluorometer and the dilution factor resulting in a concentration of ~ 100 pM was determined. The amplified libraries was enriched using the Ion PGM™ Hi-Q™ View OT2 Kit, then purified with the Ion OneTouch™ ES before loading on a 316-chip and sequenced with an Ion PGM™ Hi-Q™ View Sequencing Kit reagents. After the sequencing run, the bioinformatic analysis, such as quality and coverage analysis, alignment against the GRCh37/hg19 human reference genome  and variant calling were performed using the Torrent Suite™ Software (ThermoFisher Scientific) with the Ion Torrent Server (Dell™). The obtained Variant Caller Format (VCF) files were annotated using wANNOVAR tool (http://wannovar.wglab.org/) and compared against several databases such as the ExAC , 1000 Genomes , gnomAD . The potential functional role of the revealed mutations was predicted using PolyPhen-2  and SIFT . In order to validate the GLRA1 variations highlighted by NGS approach and in order to perform segregation analysis, DNA samples from the probands and their parents were screened using Sanger sequencing: exons 4 and 7 out of nine and exon-intron boundaries of GLRA1 gene (ref. seq.: NM_000171.3) were amplified by polymerase chain reaction using sets of oligonucleotide primers specific for exons 4 and 7 and a thermal cycler . Primer sequences and PCR conditions are available on request. PCR products were purified and directly sequenced in both forward and reverse directions on an ABI Prism 3130XL genetic analyzer  using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).Blood samples were collected from the family members, after informed consent was obtained from all of them. Genomic DNA was isolated from peripheral blood leukocytes using the salting out method. Both probands were screened using an amplicon-based gene panel that included 750 OMIM genes  the mother had the heterozygous c.895C > T or p.R299X (rs757488419), located in TM3 domain; 2) the father carried the heterozygous c.587C > A or p.D98E (rs199639315), located in ECD; 3) both probands showed these mutations in a compound heterozygous state, p.R299X inherited from the mother and p.D98E inherited from the father. Molecular variants identified are reported in Fig. https://www.ncbi.nlm.nih.gov/snp/rs199639315), was a heterozygous missense mutation in exon 4. Both PolyPhen-2  and SIFT  analysis showed that p.D98E mutation affected negatively gene function. No mutations were found in other genes known to cause familial hyperekplexia such as GLRB, SLC6A5, GPHN, and ARHGEF9.Molecular analysis of the Hyperekplexia, or Startle disease, is an uncommon non-epileptic disorder, classically characterized by exaggerated startle responses to unexpected stimuli. The abnormal startle response triggered by nose tapping is pathognomonic in hyperekplexia patients and is generally included in the examination of patients with suggestive features. In patients with hyperekplexia, no abnormalities are observed on routine blood tests, urinalysis, brain imaging studies, or EEG. Because of the overlapping clinical signs, hyperekplexia can be misdiagnosed as epilepsy, cerebral palsy, anxiety disorder, or conversion disorder . CorrectGLRA1 had been identified. Although the diagnosis of HPX is based on clinical findings, pathogenic variants in five genes have been reported to cause HPX, of which GLRA1 accounts for about 80% of cases. In fact, GLRA1 gene encoding the α1 subunit of the glycine receptor, is the major genetic cause of HPX. Dominant missense and recessive missense mutations have been identified as pathogenic variants in many individuals with the familial form of HPX and occasionally in simplex cases [GLRA1 coding region is distributed over nine exons. Hyperekplexia mutations, clustering in exons 7 and 8, induce amino acid substitutions within a region ranging from TM1 to the extracellular loop connecting segments TM2 and TM3 [GLRA1 channel function; p.D98E, in accordance with previously studies reported in literature [GLRA1 have not been reported before as compound mutations. Overall, the penetrance of HPX is 100%; however, Kwok et al. (2001), described in one family a mother who had the same variant as her two affected children and was asymptomatic [GLRA1 gene has been described before [GLRA1 are the same. It is described that parents with minor form can have children with major form or vice versa, as in our family, where mother shows a very mild form of the disease, but usually siblings tend to be affected by the same degree [GLRA1 have not been reported before as compound mutations, and that the p.R299X nonsense mutation detected in exon 7 of our patients, codifying TM3 domain, exhibited an autosomal dominant inheritance.The minor form of hyperekplexia only has excessive startle reflexes. In 2006, Bakker et al. confirmed the presence of two forms of hyperekplexia, identifying a difference in the onset, consisting in the fact that in minor form of hyperekplexia startles never begin in the neonatal period. Both forms can occur in the same pedigree [ex cases , 17. In ex cases . The GLR and TM3 . To date and TM3 . Our patptomatic . What cld before , 22. Anyd before . Howevere degree . The pree degree , 23. Dome degree , 24, 25.GLRA1 could have different phenotypic expressions, suggesting an underling mechanism of variable expressivity, even in the same pedigree. Genetic testing of glycinergic neurotransmission-associated genes, including GLRA1, is a readily available tool either to confirm clinically suspected diagnosis of HPX or for family screening. Therefore, early recognition is helpful for prompt and appropriate treatment, to avoid unnecessary investigation and may lead to genetic preconception counseling.In conclusion, our data show that the same mutation in


Failing myocytes undergo electrophysiological remodeling, which is known to be the main cause of abnormal Ca2+ homeostasis. However, structural remodeling, specifically proliferating fibroblasts coupled to myocytes in the failing heart, could also contribute to Ca2+ cycling impairment. The goal of the present study was to systematically analyze the mechanisms by which myocyte–fibroblast coupling could affect Ca2+ dynamics in normal conditions and in HF. Simulations of healthy and failing human myocytes were performed using established mathematical models, and cells were either isolated or coupled to fibroblasts. Univariate and multivariate sensitivity analyses were performed to quantify effects of ion transport pathways on biomarkers computed from intracellular [Ca2+] waveforms. Variability in ion channels and pumps was imposed and populations of models were analyzed to determine effects on Ca2+ dynamics. Our results suggest that both univariate and multivariate sensitivity analyses are valuable methodologies to shed light into the ionic mechanisms underlying Ca2+ impairment in HF, although differences between the two methodologies are observed at high parameter variability. These can result from either the fact that multivariate analyses take into account ion channels or non-linear effects of ion transport pathways on Ca2+ dynamics. Coupling either healthy or failing myocytes to fibroblasts decreased Ca2+ transients due to an indirect sink effect on action potential (AP) and thus on Ca2+ related currents. Simulations that investigated restoration of normal physiology in failing myocytes showed that Ca2+ cycling can be normalized by increasing SERCA and L-type Ca2+ current activity while decreasing Na+–Ca2+ exchange and SR Ca2+ leak. Changes required to normalize APs in failing myocytes depended on whether myocytes were coupled to fibroblasts. In conclusion, univariate and multivariate sensitivity analyses are helpful tools to understand how Ca2+ cycling is impaired in HF and how this can be exacerbated by coupling of myocytes to fibroblasts. The design of pharmacological actions to restore normal activity should take into account the degree of fibrosis in the failing heart.Heart failure (HF) is characterized by altered Ca Heart failure (HF) is a major public health problem worldwide  and the 2+ handling in myocytes, since Ca2+ homeostasis is crucial for cell contraction and relaxation (2+ transient (CaT) with a slow rise time and a reduced rate of [Ca2+]i removal that prolong CaT duration and elevate the diastolic intracellular Ca2+ level , late Na+ current (INaL), transient outward K+ current (Ito), L-type Ca2+ current (ICaL), Na+ current through the L-type channel (ICaNa), K+ current through the L-type channel (ICaK), rapid delayed rectifier K+ current (IKr), slow delayed rectifier K+ current (IKs), inward rectifier K+ current (IK1), Na+/Ca2+ exchange current (INCX), Na+/ K+ ATPase current (INaK), background currents , and sarcolemmal Ca2+ pump current (IpCa). A detailed Ca2+ dynamics is also formulated in the model. Properties such as conductances determining ionic densities and membrane kinetics can be found in the original work , INaL conductance, the maximal flux of INCX and SR Ca2+ leak (Jleak), and the fraction of active binding sites of the Ca+2 calmodulin-dependent protein kinase II (CaMKa) were upregulated, while conductances of Ito and IK1, the maximal flux of INaK, and Ca2+ uptake via SERCA pump (JSERCA), and SR Ca2+-dependence of the steady-state activation of ryanodine receptor release  were downregulated. Further details about values and experimental references can be found in Supplementary Table S2.All simulations were performed at the cellular level. To study the electrophysiological behavior of cardiac myocytes, we used the most complete undiseased human ventricular AP model, developed by nal work . We intrnal work  and leadur group . Specifigap) flows from one cell to the other through gap junctions, driven by the potential gradient between the myocyte (VM) and the fibroblast (VF) membrane potential and regulated by a junctional conductance (Ggap).The increase in fibroblasts density due to aging and/or HF, and their interaction with myocytes were modeled using established cell–cell coupling equations. Fibroblasts were resistively coupled to one myocyte (Eq. 2), and a current (I+ current (IKr), an inward-rectifying K+ current (IK1), a Na+/K+ ATPase current (INaK), and a background Na+ current . The membrane potential is governed by the following ordinary differential equation:The electrical activity of fibroblasts was formulated using an active fibroblast model . The fibgap. Regarding fibroblast properties, the membrane capacitance (CF) was set to 6.3 pF and the resting membrane potential (EF) had a value of -49.6 mV as in The electrotonic interaction with the myocyte is included in the term IM = 153.4 pF vs CF = 6.3 pF). The value of Ggap was set to 3 nS for a single fibroblast, considered the nominal value in gap fivefold , calcium transient (CaT) duration from maximal upstroke to 80% of recovery (CaTD80), CaT rise time from 10 to 90% of upstroke (t10-90), and systolic and diastolic [Ca2+]i values. All biomarkers were calculated under steady state conditions after application of 1000 stimuli at 1 Hz.To evaluate the electrophysiological activity of myocytes, and particularly CaNa, INaL, Ito, ICaL, IKr, IKs, IK1, INaK, INCX, the SR Ca2+ uptake via SERCA pump (JSERCA), the SR Ca2+ release flux via RyR (Jrel), the SR Ca2+ leak (Jleak), and INab. These parameters were varied with a scaling factor obtained from a log-normal distribution of standard deviation (σ) equal to 0.3. This led to a 95% (±2σ) of parameters varying between 55 and 182% of its control value, representing inter-individual variability and drug-induced effects. A standard deviation of 0.1 was also considered in different sets of populations.The aforementioned models were used as the baseline to generate four populations of 300 different individuals. Inter-subject electrophysiological variability was represented by variating maximal ion current conductances with a scale factor, assuming that there are variations in the number of ion channels in the cell membrane between individuals . The var90 and systolic and diastolic [Ca2+]i values, in which electrical myocyte activity was considered normal. These limits were obtained from the baseline models as APD90normal ± /2 and after being compared to experimental studies  of parameters in the range of ±60% variation was used for this purpose. In this case, only the basic HF model (with and without coupled fibroblasts) was considered. We set limits for APD studies . Table 1Linear regression was employed to analyze biomarker sensitivities to electrophysiological variables. First, a univariate sensitivity analysis was conducted varying each parameter individually by a scaling factor of equal magnitude from the baseline models. The individual variation was set to ±60 or ±15% in different sensitivity analyses. This approach was based on our previous work .R2) indicated the predictive power of the simplified linear model, being R2 = 1 a good fit.Multivariate regression analyses were then performed using the generated populations of models. We related all the conductance scaling factors configurations (x) to each biomarker (y) with regression coefficients (b) that attempted to predict the indicators (Eq. 5). These coefficients were obtained by applying partial least squares (PLS) method to data as shown in Eq. 6 and after several transformations, such as log-transformations and z-score calculations . The obtBoth types of sensitivity analyses were compared with normalized sensitivities to study the similarities and differences in parameter identification as the most important contributors to each electrophysiological characteristic. Normalized sensitivities were calculated as the ratio between each sensitivity and the maximum absolute value obtained for a particular biomarker.The comparison between isolated and coupled myocytes was performed after adjusting the bar graphs to the standard deviation of the log-transformed biomarkers in N and HF conditions, to ensure equivalent percentages of change.Figure 1 illustrates two populations of models (n = 300) for endocardial human cells paced at 1 Hz, under normal (blue traces) and HF (red traces) conditions, with baseline models for the two respective populations indicated with black solid and dashed lines. The model population calibration process, meant to reproduce natural variability, generates a wide range of physiological AP waveforms and CaTs in both groups. This variability allows us to understand predicted drug effects by considering the HF cells with behavior most similar to normal cells, or vice versa. The electrophysiological remodeling applied to the basic ORdmm model to generate a baseline HF model reproduced the characteristic HF phenotype of prolonged APD and a slower Ca2+ dynamics with depressed systolic [Ca2+]i and elevated diastolic levels. In the generated normal and failing populations, significant differences between both conditions are observed in APs and CaTs, although some traces overlap. To understand the variability within the populations and predict what alterations might cause HF cells to behave more like normal cells, we performed a sensitivity analysis.Figure 2 represents the sensitivity of quantitative indicators of AP and CaT characteristics to changes in ion channels obtained in the univariate and multivariate sensitivity analyses. Sensitivities helped reveal the most important modulators of a determined electrophysiological property. High regression coefficients highlighted an important effect of a particular transport mechanism on Ca2+ indicators, taking into account the synergy between variables in the case of multivariate analysis. Our results showed that IKr was the parameter with the major impact on APD90 , while SERCA had the leading role in Ca2+ biomarkers . Sensitivities were normalized to allow comparison between the sensitivities obtained through a ± 60% univariate sensitivity analysis (light blue for N conditions and pink bars for HF conditions) with multivariate sensitivity analysis (dark blue for N conditions and red bars for HF conditions). In this comparison, results from both methodologies were consistent for APD sensitivities . Small differences were though observed in the case of Ca2+ biomarkers . For instance, Figure 2B shows that in HF, IKr seems to have a moderate impact on CaTD80 according to the univariate results (pink bars), while multivariate sensitivity indicates that the effect is lower (red bars). In other cases, with the univariate analysis, the relative importance of parameters can be altered between the N and HF conditions. This was the case for ICaL, INCX, and Jleak on t10-90 . Figure 2D shows how NCX has the main role in modulating systolic Ca2+ according to the univariate sensitivity analysis (light blue in N and pink bars in HF), while the multivariate sensitivity highlights SERCA and ICaL as the main modulators in N (dark blue bars) and HF (red bar) conditions, respectively, and the exchanger has a secondary role.Figure 2, light and dark blue vs pink and red), such as an increased impact of INaL on APD90 in HF with respect to normal conditions , a higher IKr influence on CaTD80 , and a decrease of SERCA modulation effect on systolic [Ca2+]i .Multivariate and univariate analyses also identified similar changes in parameter sensitivities from N to HF . For example, considering low variability in HF , both analyses (univariate and multivariate) highlighted ICaL as the most important parameter regulating systolic [Ca2+]i, while univariate sensitivities obtained with 60% variability (darker pink bar) highlighted INCX as the main regulator and underestimated the impact of ICaL.We further investigated the aforementioned discrepancies by performing additional sensitivity analyses with a lower conductance variability. Parameter variability was reduced to 15% in the univariate study and the standard deviation was decreased to 0.1 in the multivariate regression, resulting in sensitivities slightly different from those described above with higher variability . After reducing parameter variability, negligible differences were found when comparing both methods . This indicates that, with large parameter variability, and in the HF condition, significant non-linear relationships between parameter values and Ca2+ handling processes make the multivariable regression model less accurate.Coefficients of determination of the multivariable regression are shown in In summary, univariate and multivariate sensitivity analyses yield very similar results for low variability of parameters, which would correspond to natural inter-subject electrophysiological differences. Univariate analysis is less computational expensive and is thus a valid methodology under these conditions. However, when large variability is applied, which would respond to effects of drugs, significant discrepancies arise between both methodologies. Multivariate analysis should be more reliable, at least for biomarkers with coefficients of determination close to one. Indeed, multivariate analysis considers interaction between the different parameters of the ion channels altered.Figure 3 shows APD and systolic [Ca2+]i reduction in the normal baseline human endocardial model when fibroblasts were coupled (solid vs discontinuous blue trace), as well as in the failing baseline endocardial model (solid vs discontinuous red trace).When simulations were performed to electrically coupled fibroblasts to a myocyte, the myocyte’s AP and CaT were significantly changed. n = 300) generated for both N and HF conditions, the above-mentioned effects on APD and CaT were maintained, as shown in Figure 4. CaT traces in normal conditions  with coupled fibroblasts (discontinuous blue traces) overlapped with CaT traces in the absence of fibroblasts (solid blue traces).When fibroblasts were coupled to myocytes from two populations of models  decreased both in normal and failing conditions when fibroblasts were considered (discontinuous bars).To evaluate whether fibroblasts coupling would significantly change sensitivities of electrophysiological biomarkers to parameters variability, multivariate analyses were conducted on these new populations. When a high variability of parameters was considered (σ = 0.3), our results showed that in the presence of fibroblasts, biomarkers sensitivities to ionic variables slightly changed with respect to uncoupled myocytes. As displayed in Supplementary Figure S3), sensitivities hardly changed with respect to higher variability.To confirm these results, we decreased parameter variability to σ = 0.1 and obtained more accurate regression coefficients. In the new range of parameter variability . The population of models approach generates cells exhibiting a wide variety of physiological behaviors, including cells within the HF population that have APs and CaTs similar to those observed in healthy cells. Selecting this subpopulation and examining the distributions of parameters provides guidance for therapeutic targets. We identified parameter combinations restoring APs and CaTs to waveforms within a healthy range shown in Table 1 in Section “Materials and Methods.” In Figure 6A, red traces represent all models generated from the baseline ORdmm HF model (black dashed line) and blue lines are models satisfying healthy ranges of both AP and CaT biomarkers. Electrophysiological HF phenotype was restored in around 500 models when IKr, SERCA, and ICaL activities were enhanced, and NCX function decreased. Interestingly, NCX distribution presented several outliers at high scaling factors. When myocytes were coupled to fibroblasts , the role of SERCA, ICaL, and NCX was similar, whereas IKr modulation was not important.To analyze how failing APs and CaTs could be restored and brought to normal ranges, a larger population of failing models was generated  and without (dashed line) fibroblast coupling. To understand how these steady-state conditions were reached, we analyzed transient changes in myocyte [Ca2+] after fibroblast coupling, as shown in Figure 8. In these panels, isolated myocytes are initially at steady state, then fibroblasts are coupled beginning on the second beat. As shown in Figure 7C, Igap is an outward current flowing from the myocyte to fibroblasts, non-existing in the uncoupled myocyte. This outward current decreases membrane potential and has an indirect effect on voltage dependent currents such as ICaL. Indeed, we can observe that when fibroblasts are coupled (dashed traces) all currents and fluxes present a shorter duration, as well as AP, and a reduced peak , except ICaL. Despite the reduced duration of this current, the initial ICaL peak contributed to a larger Ca2+ influx through these channels (increase from 140 to 146 pC/μF). The integral of NCX was also computed and indicated an increase in inward NCX extruding more Ca2+ when fibroblasts were coupled (from 77 to 81 pC/μF). In the transient evolution shown in Figure 8F, the peak of ICaL exhibited a maintained increase compared with the uncoupled myocyte (shown in solid red). Due to the increased Ca2+ extrusion through NCX, however, [Ca2+]JSR progressively declined, leading to the decrease in [Ca2+]i peak observed in steady state.Fibroblast coupling decreased systolic Ca2+ impairment in HF, fibroblast to myocyte coupling does not alter the role of the main mechanisms regulating Ca2+ dynamics in myocytes, and (iii) drug action on ICaL and SERCA enhancement and NCX block would help CaT restoration in HF regardless of fibrosis presence.In this study, two methodologies were used to perform sensitivity analyses evaluating the effects of fibroblast–myocyte coupling under normal and HF conditions. Our main findings are that (i) univariate and multivariate analyses yield very similar results and low variability of parameters yields more reliable multivariate sensitivity analyses, (ii) despite exacerbating CaNaL, or the decrease of systolic [Ca2+]i sensitivity to SERCA in HF with respect to N conditions.Univariate and multivariate sensitivity analyses were performed using an initial large variability (±60%) and then smaller (±15%). At lower variability, changes in electrophysiological properties behaved linearly, making results from univariate and multivariate analysis more similar. Indeed, ionic currents and fluxes work synergistically to generate AP and CaT. Variables such as membrane potential and ionic concentrations link all ionic mechanisms in a way that a change in one parameter (conductivity in this case) has an effect on other parameters, and final differences in electrophysiological properties are the result of an interaction of all these variations. In addition, within the range of variation of a specific parameter, the effects on biomarkers can be different, and assuming linearity can become less accurate when a wide interval is considered. In fact, as electrophysiological remodeling in HF is simulated by changing parameters, the same scale factor applied in N and HF implies a different range of variation of such parameters, accounting for different sensitivities to biomarkers. This explains the increase in APD sensitivity to IMultivariate parameter sets, unlike varying one parameter at a time, can provide information about electrophysiological properties in a wide combination of parameters, which could be useful to evaluate the response of a drug in different individuals, instead of on a fixed baseline model. The high physiological or pathological variability of ionic parameters requires the analysis of the behavior in the whole range.2+ dynamics. We observed that univariate and multivariate sensitivities agreed in the most important parameters contributing to each biomarker. For instance, it was observed the strong impact of IKr on APD90, as CaL was highlighted with both methodologies, agreeing with experimental observations  changing to myofibroblast phenotype , and we observed similar effects, i.e., APD shortening and Ca2+-handling impairment. The improvement of myofibroblast models, incorporating Ca2+ dynamics, would certainly shed light into the understanding of Ca2+ dynamics alterations in the failing tissue.An important effect observed when simulating fibroblast–myocyte coupling is the marked APD shortening. Experimental studies showing the existence of gap junctions (connexin43) between fibroblasts and myocytes also revealed changes in AP waveform and conduction velocity due to a modulation of myocyte electrophysiology . Previou cycling . To date cycling . In the in vivo coupling, different degrees of fibrosis, and the difficult to quantify coupled fibroblasts in tissue has led to the exploration of different values of Ggap or a range of myocyte–fibroblast ratios , we observed that ICaL peak increased since the first beat, due to the immediate effects of Igap on AP. Initially, there was also an intracellular Ca2+ rise, as SR Ca2+ content was still elevated. The mechanisms to remove Ca2+ from the cytosol were then activated: SERCA and inward current through NCX (extruding Ca2+). NCX role, extruding Ca2+ out of the cell, becomes relevant in the transient state because if there is an imbalance with Ca2+ influx through ICaL channels, Ca2+ loss occurs. This mechanism might explain the reduced SR Ca2+ load in myocytes interacting with fibroblasts.Our findings showed that a higher Iller CaT . Therefo2+ impairment in HF by further reducing SR Ca2+ content in myocytes.Our findings suggest that fibroblasts increase Ca2+ dynamics in these cells. Ca2+ signaling has been observed in human cardiac fibroblasts although with different pathways to those in contractile myocytes (in vivo.Several limitations need to be considered when drawing conclusions from the present study. Although simulations of cellular electrophysiological behavior and systematic analyses of biomarkers complement and enrich experimental research, uncertainties in the development of the mathematical models employed might affect the outcome of the simulations. The limited availability of electrophysiology data from human fibroblasts has led to the use of a fibroblast model developed on the basis of adult rat ventricular tissue data, without taking into consideration potential changes in HF. Fibroblasts with the same characteristics were considered in normal and failing conditions, but electrophysiological remodeling in fibroblast currents could have an additional effect on myocytes . Anothermyocytes , and myomyocytes , but nei2+ can modulate the coupling current. If the modulation of the intercellular coupling by Ca2+ ions was taken into account, larger alterations in Ca2+ dynamics mechanisms could be observed. Any mechanism related to Ca2+ included into our model, such as realistic Ca2+ dynamics in fibroblasts, may affect the regulation of Ca2+ cycling in myocytes, and enhance the influence of fibroblasts.Gap junctional coupling between a myocyte and a fibroblast has been modeled by a simple conductance as in previous simulation studies of fibroblast-myocyte coupling. However, recent findings suggesting the interaction of connexin45 with CaM  highligh2+ release resulting from detubulation. However, a detailed model including local changes in membrane structure related to the spatial organization of ion channels in HF could provide more accurate results about Ca2+ impairment (Other structural modifications in HF, concerning myocytes, involve the loss of transverse tubules and reorganization of the cell membrane . We havepairment .2+ homeostasis is related to contraction and relaxation force, other mechanical factors, such as myofibroblast contractility when fibroblasts are differentiated into the activated form or extracellular stiffness due to the excessive accumulation of collagen in HF, can contribute to cardiac dysfunction. Our model only considers intracellular Ca2+ in myocytes at a cellular level to evaluate the contraction of the heart. The behavior in tissue or even in the whole organ could also be different. A defined architecture of the myocyte when considering spatial distribution would allow to take into consideration the distribution of gap junctions as well as the extracellular space which could modulate Ca2+ homeostasis as it does in electrical propagation (Although Capagation . We conspagation .Regarding sensitivity analyses, the non-linearities are the main issue in quantifying the impact of parameters on biomarkers. To restore the electrical activity and contraction of myocytes, the effect of some drugs can imply a high change in an ionic mechanism, and the predictive value of this methodology decreases with increased variability.2+ impairment observed in HF, which together with the arrhythmogenic activity of fibrotic tissue, can compromise the function of the myocardium. In the present study, the measures suggested to restore Ca2+ dynamics and contractility at cellular level are reliable because they are supported by experiments and according to our findings, tackling the electrophysiology remodeling in failing myocytes can also improve the effects of fibroblasts interactions.Nonetheless, despite these limitations, cellular simulations can shed light in the causes of CaMM, JF, and BT conceived and designed the study, and analyzed the data. MM performed simulations. JG and ES contributed to the methodology. MM and BT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Serum and urinary levels of SLPI were quantified by ELISA. SLPI was significantly elevated in AKI patients compared with non-AKI patients . The area under the receiver operating characteristic curve of serum SLPI 6 h after surgery was 0.87 ((0.76–0.97); DC). The addition of SLPI to standard clinical predictors significantly improved the predictive accuracy of AKI  = 3.91 (1.44–12.13)). In a subgroup, the increase in serum SLPI was evident before AKI was diagnosed on the basis of serum creatinine or urine output ). In this study, SLPI was identified as a novel candidate biomarker for the early diagnosis of AKI after cardiac surgery.Acute kidney injury (AKI) is one of the most frequent complications after cardiac surgery and is associated with poor outcomes. Biomarkers of AKI are crucial for the early diagnosis of this condition. Secretory leukocyte protease inhibitor (SLPI) is an alarm anti-protease that has been implicated in the pathogenesis of AKI but has not yet been studied as a diagnostic biomarker of AKI. Using two independent cohorts (development cohort (DC),  Acute kidney injury (AKI) is one of the most common complications after major surgery, especially after cardiac surgery ,2. DespiAccordingly, studies have investigated several new biomarkers of AKI, and urinary tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) ([TIMP-2]·[IGFBP7]) have been deemed promising markers . The assThe aim of the study was to investigate the association between serum SLPI levels and the incidence of postoperative AKI after cardiac surgery. The studies, registered at clinicaltrials.gov , were approved by the institutional review board  and performed in adherence to the Declaration of Helsinki. All patients were scheduled for elective cardiac surgery involving aortic cross-clamping, cardioplegic myocardial arrest, and cardiopulmonary bypass. Exclusion criteria were emergency operations, pregnancy, lack of informed consent, an age of less than 18 years, and end-stage renal disease requiring dialysis. First, an explorative development study was conducted between September 2015 and March 2016. Second, the results of the development study were used as the basis for a prospective observational validation study, which was conducted from January to June 2017.Serum creatinine was measured daily. The serum creatinine level on the day before cardiac surgery was used as the reference value. Urine output was quantified hourly by Foley catheter drainage while the patient remained in the intensive care unit.In both the development and validation studies, serum samples for enzyme-linked immunosorbent assays (ELISA) were drawn one day prior to surgery and immediately (0 h) and 24 h after surgery. In the development study, additional samples were drawn 6 and 12 h after surgery, and, in the validation study, an additional sample was collected 48 h after surgery B.After blood collection, the samples were centrifuged (3000 rpm for 10 min), and the supernatants were transferred to cryotubes for storage at −80 °C until further analysis. Urine samples were collected preoperatively, immediately postoperatively, and 24 h after surgery and transferred to cryotubes for storage at −80 °C.The primary endpoint of both studies was the development of AKI within 72 h after cardiac surgery. AKI was diagnosed according to the KDIGO clinical practice guidelines by (1) an increase in serum creatinine of at least 0.3 mg/dL or a 50% increase from baseline and/or (2) a decline in urine output to below 0.5 mL/kg/h for at least 6 h .The following patients’ baseline characteristics are known to affect the risk of AKI and were thus determined: age, sex, body mass index (BMI), intake of heart medication, arterial hypertension, pulmonary hypertension, congestive heart disease, reduced left ventricular ejection fraction (LVEF) <35%, chronic kidney disease, chronic obstructive pulmonary disease (COPD), diabetes, and previous cardiac surgery. On the basis of these data, we calculated the Cleveland Clinic Foundation Score—a clinical score used to estimate the risk of developing AKI after cardiac surgery . We recoSerum and urine levels of SLPI were measured by ELISA as previously described and according to the manufacturer’s instructions  . For theBecause SLPI serum levels ranged widely in the first study (development study), we performed an additional validation study. The sample size of the validation study was calculated on the basis of the development study. The median of the SLPI levels 24 h after surgery was used as a cut-off. We assumed a power of 90% and set the significance level to 0.05. From the difference in proportions of the “AKI events” in both groups (G1 (≤median SLPI) = 10.7% and G2 (>median SLPI) = 34.5%), we calculated a preferable sample size of 168 patients, assuming a drop-out rate of 25%. The sample size was calculated with PROC POWER, SAS 9.4 .Categorical variables are described by absolute numbers and percentages, and continuous variables are reported as the median and interquartile range (IQR) with the first (Q1) and third (Q3) quartiles.Differences in baseline characteristics between the two outcome groups were analyzed using univariable logistic regression. Correlations between SLPI and NGAL were calculated using Spearman’s correlation.p-values were calculated for a hypothesis of AUC > 0.5. Optimal cut-offs were calculated using the Youden index, and 95% confidence intervals, sensitivities, and specificities are reported.The diagnostic accuracy of the biomarkers was calculated by the receiver operating characteristic (ROC) curve and the corresponding area under the curve (AUC). A univariable logistic regression model was used to investigate the performance of SLPI as a predictor of AKI. Given a nonlinear relationship between SLPI and the incidence of AKI, SLPI was considered a binary variable and categorized by the corresponding median to avoid biased estimates .P-values are reported. To evaluate whether serum SLPI is able to predict AKI before an increase in serum creatinine is evident, we analyzed the time point at which a rise in serum creatinine was detected ·[IGFBP7] have provided promising results ,19,45. TIn conclusion, we identified SLPI as a novel biomarker for the early detection of AKI after cardiac surgery. Our findings may lead to future perspectives of biomarker-based risk stratification and may identify patients who would benefit from the early initiation of preventive and treatment strategies.