Banana is a fruit grown mainly in tropical countries of the world. After harvest, almost 60% of banana biomass is left as waste. Worldwide, about 114.08 million metric tons of banana waste-loss are produced, leading to environmental problems such as the excessive emission of greenhouse gases. These wastes contain a high content of paramount industrial importance, such as cellulose, hemicellulose and natural fibers that various processes can modify, such as bacterial fermentation and anaerobic degradation, to obtain bioplastics, organic fertilizers and biofuels such as ethanol, biogas, hydrogen and biodiesel. In addition, they can be used in wastewater treatment methods by producing low-cost biofilters and obtaining activated carbon from rachis and banana peel. Furthermore, nanometric fibers commonly used in nanotechnology applications and silver nanoparticles useful in therapeutic cancer treatments, can be produced from banana pseudostems. The review aims to demonstrate the contribution of the recovery of banana production waste-loss towards a circular economy that would boost the economy of Latin America and many other countries of emerging economies. The food sector is divided into different industries, such as the pasta industry, as well as fruits and vegetables, fish, meat and beverages , transfoFruits and vegetables are vital in the daily diet of humans, whose demand is constantly growing due to the increase in population and changes in eating habits. According to the Food and Agriculture Organization of the United Nations (FAO), the world trade of mainly tropical fruits reached a maximum of 7.7 million tons in 2019, an increase of 6.4% , compared to the previous year [Banana is a tropical fruit grown in more than 130 countries. It is the second most-produced fruit after citrus, contributing around 16% of world fruit production and the fourth most important food crop after rice, wheat and corn. Banana is very nutritious and digests better than many other fruits. Their vast consumption is due to their sensory characteristics and attractive texture and flavor. In addition, it has a high calorie content, with a small amount of fat, and is an excellent source of dietary fiber, vitamin C, vitamin B6 and manganese .Diets rich in fruits and vegetables with phenols are associated with a lower risk of cancer and heart disease . PhenoliIn 2019, India led banana production with 30.4 million tons, followed by China with 11.6 million tons . The thiAccording to the United Nations Food and Agriculture Organization (2020) report, banana production had significant growth in the last few years. The market analysis determined that exports reached 20.2 million tons in 2019, due to the growth of banana production in Ecuador and the Philippines, with an estimated growth for 2028 of 135 million tons [According to data from the Ministry of Agriculture and Livestock, total exports of bananas and their products in Ecuador from January to July 2019 were worth two billion dollars, representing 51% of the agricultural production .In 2019, 51,227 hectares of bananas were planted in Colombia. Colombian banana exports were estimated at USD 852.8 million, while those exported to Belgium were worth USD 204.8 million and those to the United Kingdom were worth USD 169.6 million. About USD 149 million-worth were exported to the United States, and USD 124.8 million-worth to Italy .Several countries produced and exported bananas in Europe in 2019, such as the Netherlands, Belgium and Germany, with revenues of USD 790.6 million, USD 782.1 million and USD 242.1 million, respectively .In Asia, banana exports reached a maximum of 5.1 million tons, representing an increase of 42% compared to the 3.5 million tons exported in 2018 . The PhiThe leading sales from the banana industry derive from fresh banana sales, while the processing rate is less than 20%. Therefore, banana processing has excellent development potential due to the generation and use of processing waste-loss in new production chains. The advanced processing technology is the driving force to improve the added value of commodities and the resource utilization rate of bananas .The banana industry during its production cycle generates large volumes of solid waste-loss derived from maintenance and harvesting processes, highlighting the rachis, pseudostems, leaves and banana peel, which can be used in different processes, such as packaging products and other applications based on bioplastics, thus contributing to the implementation of a circular economy .There are different studies on the processing of bananas and the application of their waste-loss in new production processes ,19,20,21The circular economy (CE) can be defined as an economic model based on the efficient use of resources through the minimization of waste, long-term value retention, reduction of primary resources and closed production cycles within the limits of socio-environmental policies . The traThe internal circles of production, reuse and reconditioning demand fewer resources and energy and are more economical than conventional recycling . ClimateAn additional benefit of applying a circular economy would help organizations hedge against volatility in commodity prices and rebalance flows of goods, scrap and used products . The EllThe review aims to demonstrate the contribution of banana production waste-loss towards the implementation of the circular economy by proposing several possible applications  in order to provide the reader with innovative, sustainable and low-cost ideas that will allow relief in the global economy.Banana is a large-scale, evergreen herbaceous plant belonging to the Zingiberales order, Musaceae family and Musa genus, with high calcium, magnesium and high assimilation of nitrogen emissions . The banIn general, bananas have more than 50 species and dozens of hybrids, large and rhizomatous underground stems, from which their large leaves with powerfully spirally arranged pods start, giving the shape of a false stem  and Musa balbisiana Colla (genome B) [It belongs to the Musaceae family, order Scitamineae. This family is made up of the enome B) . Currentenome B) . To illuBanana contains sugars, acids, vitamin C, amino acids and pectin that form the fruit’s soluble solids content and vary according to the variety grown and the degree of maturity . During The fruit contains various chemical components , especiaThe peel and pulp are good sources of certain biogenic amines (catecholamines) produced by decarboxylation of amino acids or amination of aldehydes and ketones. Catecholamines include dopamine, serotonin, epinephrine and norepinephrine . SerotonBananas, like most fruits, are acidic, with a pulp pH below 4.5. The primary organic acids in bananas are ascorbic, citric, malic and oxalic acid, industrially essential acids because they mainly act as antimicrobial agents, neutralizers and preservatives for food .Bananas are considered a household fruit and are mainly characterized by their fresh consumption. However, several finished products are derived from the processing and handling fresh bananas and their components . Banana Export bananas are subjected to an intensive quality control process to ensure that they reach the final destination in an optimal state of ripeness and free of stains, dirt and scars from abuse . At the The banana industry is characterized by producing waste-loss from roots, stems, leaves, or any other part of the plant  that is Banana peel is one of the essential waste-loss generated from processing. Bananas contain 60% pulp and 40% peel, 7.25 kg of peel produced from a banana box of 18.14 kg . HoweverPseudostems are the banana stem that supplies nutrients from the soil to the fruits. It is structured by two elements, the nodes and internodes, which make up the floral stem and support the inflorescence in the inner part . The outBanana pulp is a rich source of essential phytonutrients, including phenolic compounds and vitamins . It also contains carotenoids, flavonoids, amine compounds and dietary fiber . DietaryIn recent decades, the world population increased up to 7 billion, generating around 683 million tons of agri-food waste, 34% related to waste-loss of food produced worldwide .The use of this waste-loss has become a topic of great interest, leading to the transition and implementation of a circular economy model as a regenerative economic model . The objCavendish and Gros Michel varieties, due to their high availability in different countries such as India, Philippines and Ecuador, detailed in Musa paradisiaca spp. is one of the most demanded varieties in the harvesting industry. Thanks to the composition of its residues, it can be used in all the applications mentioned above. Banana Cavendish is widely used in biofuels because sugars represent the species’ main component of soluble solids. By subjecting them to cellulolytic organisms, a greater quantity of fermented sugars is necessary for biofuel production. Furthermore, the varieties Williams, Musa balbisiana and Musa accuminata have good potential for use in wastewater treatment, bioplastics and nanotechnology applications thanks to their physicochemical properties, such as its lignocellulosic content, its density and its ability to adapt to extreme conditions of climate, soil and water. The chemical composition and physical structure of the waste-loss of banana studied in this paper can be observed in the different tables in Sustainable development of the circular economy concept is only possible if technologies are adopted to recover waste-losses. The recovery of food waste-loss opens up new economic growth horizons, allowing the opportunity to transform raw material into a circular loop . The banDue to the rapid development of the automotive industry and environmental pollution problems, the growing demand for petroleum-derived fuel has inspired efforts to explore alternative fuels .The lignocellulosic biomass from the shell, pseudostems and rachis of banana represents a source of promising raw material for ethanol production due to its abundance and high availability. Agri-food waste can be used locally, and does not trigger competition between fuel and food . The proSaccharomyces cerevisiae [v/v) [The banana rachis is chopped to reduce their size to 1 cm in width in the pretreatment, dried and later crushed for their correct use in the other processes . The enzrevisiae . Finallyae [v/v) .v/v) [Finally, with the distillation of the alcohol, the aim is to remove the final product due to the difference in boiling point, and as much water is separated to obtain alcoholic beverages with the highest concentration possible (around 40% v/v) .On the other hand, the fermentation of banana waste-loss can produce biogas. The production occurs through anaerobic fermentation, one of the most frequent biological processes used to decompose organic materials . Biogas 2O5 catalysts promoted with Pd and Ru has received particular attention in research, since it shows promising selectivity for hydrogen selectivity and ethanol conversion in reform reactions using low-cost materials [Another biofuel alternative that is produced from the use of banana waste-loss is hydrogen. Biomass gasification is a promising technology for converting different raw materials for various energy purposes. This complex thermochemical process converts lignocellulosic materials into a more valuable gas known as synthesis gas through a series of reactions at high temperatures, with the presence of gasifying agents such as air, steam, oxygen or mixtures . The proaterials . Hydrogeaterials .Biodiesel is produced from the transesterification of a wide variety of oils and fats (triglycerides), including recycled oils and low-molecular-weight alcohols (mainly methanol and ethanol) . HoweverAccording to the World Wildlife Organization (WWF), more than 1100 million people do not have access to drinking water worldwide because the growing population demands safe drinking water insurance .Several organic and inorganic products are considered toxic pollutants frequently released into the environment, mainly in surface waters . The indThere is a progressive increase in heavy metals such as chromium, mercury, nickel, cadmium and copper in drinking and wastewater samples that account for the increasing contamination. In the case of hexavalent chromium, it behaves as a toxic element. Chromium is considered harmful for red blood cells, liver, spleen, kidney, soft tissues and bones, affecting the digestive, respiratory and urinary systems . ChromiuBanana rachis has been used in adsorption and chemical precipitation methods to remove chromium from wastewater, obtaining a removal efficiency of 99.8% at pH 6.7 . Banana On the other hand, lead, a reasonably malleable corrosion-resistant metal, because it melts quickly, has several uses, and is expected mainly in paints and toxic fumes from industries such as metallurgy, mining and construction industries . It has Another pollutant in wastewater is cadmium, formed by burning metals in the air or pyrolysis of carbonates and nitrates. Cadmium toxicity can affect bones, the respiratory system, kidneys, reproduction and is potentially carcinogenic . SometimMusa paradisiaca have been treated with chemical compounds, such as HCl and NaOH, to obtain activated carbon useful in the chromium removal from wastewater produced in the textile industry [Several studies have been reported on the treatment of wastewater using banana waste-loss . Celluloindustry .Polymers can be natural, synthetic or semi-synthetic . NaturalBioplastics are also produced using other polysaccharides such as polyhydroxyalkanoates, collagen and lipids from vegetable oils. In this sense, food waste-loss as an initial raw material is a good option for elaborating biodegradable materials . Starch The peels must be properly washed, cut and then heated with water to produce bioplastics from banana starch. Then they must be crushed and mixed with compounds such as acetic acid, glycerol, or sodium hydroxide . The mixBacillus megaterium, Methylobacterium rhodesianum, Alcaligenes eutrophus, Methylorubrum extorquens, Pseudomonas putida, Sphaerotilus natans and Escherichia coli, among others, are known as producers of PHB [Polyhydroxybutyrate (PHB) is a biodegradable thermoplastic synthesized by submerged fermentation from renewable raw materials. It is a polyester composed of highly crystalline, rigid and brittle linear elastomers with chemical resistance to hydrolysis . PHB hass of PHB . Throughs of PHB . Due to s of PHB .On the other hand, film food packaging avoids the loss of water, which causes a modification in texture and rigidity. In addition, food-packaging delays chemical changes such as color, aroma and nutritional value, since they act as a barrier against gases or volatile loss compounds that influences chemical and microbiological stability and avoid food decay and loss . Banana Nanotechnology is a field of vertiginous development in recent years, thanks to the multiple applications in medicine, food, cosmetics and pharmaceuticals, among other fields . The maiMusaceae waste-losses have been reported, especially cellulose production  when the organic substrate  is polymerized during cultivation [Another alternative to produce nanocellulose is through the use of microorganisms such as tivation ,156.Gluconacetobacter xylinus in fermentation to produce water filter membranes, the synthesis of bacterial nanocellulose (BNC) from Musa paradisiaca (w/v) [Komagataeibacter medellinensis and 26.4 g/L of glucose, 16 g of BNC was obtained after 12 days of fermentation in a plastic bioreactor in Colombia [Using the bacterium adisiaca  was achica (w/v) . AdditioColombia . BNC appColombia . The proColombia , and alsColombia .Saccharomyces cerevisiae and Cryptococcus sp. makes it possible to produce oleaginous and indigenous yeast with applications in ethanol production and biodiesel, respectively [Saccharomyces sp. and fermented sugar cane inulase can be synthesized using banana peel as substrate. Furthermore, using banana residues as a carbon source for Bacillus subtilis, it is possible to obtain alpha-amylase [Banana residues are not only used to feed microorganisms that produce BNC. This mechanism is also used in various recovery processes, such as obtaining biofuels and producing enzymes. For example, it has been proven that using banana peel as an energy source for ectively ,162. It -amylase .Commercially available silver nanoparticles are synthesized through wet and dry synthesis procedures and approved in consumer products. Many methods have been reported to synthesize silver nanoparticles using chemical, physical, photochemical, green and biological routes . The che3 to be reduced and form Ag0, which is stabilized by starch or some other polysaccharides shown in 3, HCl, NaOH and FeCl3, for a subsequent Soxhlet-type extraction, then filtered to remove impurities and stored in a refrigerator [Due to their reductive properties, banana leaves can be used in the organic synthesis of silver nanoparticles. The reducing sugars glucose, fructose and xylose allow Ag+ from the AgNOigerator . Interesigerator .Silver nanoparticles have great utility due to their different properties, thanks to their size, optical, magnetic and electrical shape, usually used for coating materials, biosensors, antimicrobial applications, electronic components and composite fibers .Williams banana peel has been used to obtain nano fertilizers, obtaining sizes in the range of 19 to 59 nm  involves the biodegradation of organic matter by worms to maintain the flow of nutrients from one system to another . There aDecomposition begins in the gizzards of earthworms, where organic matter is digested. Earthworms can accumulate heavy metals, contaminating organisms and increasing microorganisms’ resource availability by crushing the waste into smaller particles . OrganicWhen used as a fertilizer, VC positively impacts soil quality, plant growth, crop yield and nutritional value. The use of VC in the soil also improves physicochemical characteristics  . LikewisThe banana industry generates a large amount of waste-loss that can be used in various recovery processes, such as biofuels, wastewater treatment and the production of bioplastics focused on implementing a circular economy. Due to its high content of organic compounds rich in carbon such as polysaccharides, banana peel has been used mainly to obtain bioplastics with high degradation rates, and produce biofuels such as diesel and ethanol. The leaves from the banana plant are used to produce biodegradable packaging and utensils and organic fertilizers. Due to their fibrous composition, the banana’s pseudostems are used to obtain bioplastics requiring little time for biodegradation. Cellulose and hemicellulose banana waste-loss content is of great importance in nanotechnological processes, especially in producing green nanoparticles. It has been shown that the leaves, rachis, pseudostems and banana peel have a high potential for use, which can be reused in various recovery processes. In addition, it allows to close production cycles and reduce the accumulation of waste from the banana industry through its use in different applications, contributing to the growth of the circular economy.Despite the wide range of application possibilities for banana industry waste, production techniques must be improved to obtain products with better characteristics, such as improving the biofuel process’s pre-treatment technique to produce more energy-efficient end products. However, the lack of pilot-scale studies and the industrial implementation of these wastes is evident, suggesting the need for more studies at higher-scale applications. We cannot forget that companies seek to innovate their productive activities to obtain products with added value every day. For that reason, it is necessary to implement the various possibilities exhibited in this review to add value to the banana industry in a sustainable way, contributing to a circular economy.


APTES also played the role of a coupling agent. The use ofadvanced techniques  performed by means of a multirange device Ganesha 300XL+) allowed us to evidence a multisheet structure of the nanoparticlesinstead of the gel one typically obtained through a sol–gelroute. A mechanism combining in a new manner well-assessed knowledgeregarding sol–gel chemistry, emulsion formation, and Ostwaldripening allowed us to give an explanation for the formation of theobserved lamellar nanoparticles.Organic/inorganichybrid composite materials with the dispersedphases in sizes down to a few tens of nanometers raised very greatinterest. In this paper, it is shown that silica/epoxy nanocompositeswith a silica content of 6 wt % may be obtained with an “ Very often, unprecedentedmaterials were produced whose properties are not simply a combinationof the two components alone.15 A commonway to develop these materials is the use of the sol–gel synthesisroute.19 The mild synthesis conditions in the sol–gel process allowgenerating an inorganic phase in the presence of the monomer or thepolymer through the so-called “in situ”process, in which both phases are formed successively in one procedure.20 This is of great interest and was recently actively explored alsoin the case of non silicatic fillers.22 When dealingwith sol–gel chemistry, if the reaction rates are comparable,an interpenetrating network may also be obtained from the simultaneousreactions of the organic monomer and the sol–gel precursor.20 In addition, the sol–gel route offersthe formation of an inorganic phase starting from simple low-molecular-weightprecursors, allowing a nanolevel molecular design of the final structure.This “bottom-up” approach is necessary for HOI production.15 It is also worth mentioning here that the HOI development took advantageof the availability of instruments for nanoscale observation and characterization,in particular high-resolution transmission electron microscopy (HRTEM)and small-angle X-ray scattering (SAXS). One of the most studied HOIsystems is based on epoxy/silica, which is characterized by silicananoparticles well dispersed in a cured epoxy matrix.6 Recently, the authors exploited the sol–gel chemistry toproduce a silica/epoxy HOI with an inorganic content up to 6 wt %.26 Two silica precursors were used: tetraethyl orthosilicate (TEOS)and (3-aminopropyl)-triethoxysilane (APTES). APTES also played therole of a coupling agent between silica and the epoxy matrix by allowingchemical reactions between the components. The molar ratio TEOS/APTESwas kept constant (2.3:1) in these studies. The experimental results proved thatthe dispersed phase consisted of silica particles, a few nanometersin size, well dispersed in the epoxy network. Despite the very lowsilica content, the HOI exhibited an excellent fire behavior: absenceof dripping during a vertical flame spread test, formation of a continuousand stable char, and a reduction in the heat release rate (HRR), downto 60% of the neat epoxy resin one.26Recently,very great interest has been devoted to the developmentof organic/inorganic hybrid (HOI) composite materials with the dispersedphases in these materials in sizes down to a few tens of nanometers.26 allows us to prepare silica/epoxy composites with a silica contentof 6 wt % and TEOS/APTES molar ratio as low as 1.25. The structureof the silica nanoparticles formed is studied with the aid of bright-fieldHRTEM as well as X-ray scattering over an unprecedented q range .33 The hydrolysis and polycondensation reactions in the case of thevery popular alkoxide precursors Si(OR)4 can be writtenas33In this work, it is shownthat the procedure employed, which followedprevious works,Tetraethylorthosilicate , (3-aminopropyl)-triethoxysilane, and ethanol  were purchasedfrom Sigma-Aldrich (Switzerland). A two-component epoxy resin system, consisting of bisphenol A diglycidylether (DGEBA) and modified cycloaliphatic polyamines, was used forfabricating composite laminates.26 Thus, an “in situ” sol–gel synthesis was promoted prior to the additionof the epoxy hardener. The TEOS/APTES weight ratio was kept equalto 1.25. So as indicated in the second step reported below, silicaformation required a higher temperature (80 °C instead of roomtemperature) and reflux conditions.34 Thesynthesis was performed in one pot involving the following three steps:A mixture of epoxy (DGEBA) and APTESwith a weight ratioof epoxy/APTES of 100/5 was stirred vigorously at 80 °C for 2h to get a silanized epoxy.TEOS, distilledwater, and ethanol (EtOH) were addedto the silanized epoxy and subsequently stirred vigorously at 80 °Cunder reflux for 90 min, and the reaction vessel was then opened andkept at 80 °C for 30 min in order to remove ethanol and water.The amount of hardener needed for the curingwas thenadded to the mixture at room temperature and mixed for 5 min.The resulting mixture was degassed undervacuum andpoured into a Teflon mold. The curing process was carried out at 30°C for 24 h; then, the sample was post-cured at 80 °C for4 h.APTES and TEOSwere used as silica precursors and added to the commercial two-componentepoxy resin system by following the same synthesis route reportedin the literature.EPO6Si_1.25  throughout the paper.The silica content estimated fromthe stoichiometrywas 6 wt %, and the hybrid sample was coded as –1 and 32 scans and a Thermo Scientific OMNIC Software Suite . All the obtained spectra werenormalized to the strong absorption bands at 1607 and 1509 cm–1, related to the bonds of the benzene rings presentin the epoxy resin structure, that are not expected to change afterthe curing reaction.FTIR transmittancespectra were recorded with a Nicolet 5700 FTIR spectrometer , using a single-reflection attenuated totalreflectance (ATR) accessory with a resolution of 4 cmDMAtests were carriedout on a DMA3300 (TA Instruments). The tests were run in a three-pointbending mode with a span of 40 mm and a frequency of 1 Hz; the widthof samples was about 10 mm and the temperature was ramped from 25to 100 °C at a heating rate of 3 K/min. The analysis was repeatedon three samples of each composition.TEM images of compositesamples were recorded using a TEM/STEM JEOL JEM 2200 fs microscopeoperating at 200 kV. Prior to TEM analysis, powders of the samplewere prepared and dispersed in water and a drop of finely dispersedsample was put on a Lacey Carbon film copper TEM grid. The TEM gridwith the sample droplet was dried overnight in an oven at 40 °C.50 particles at random locations were analyzed by Image J to determinethe particle size and distribution. HRTEM images were used to determinethe lattice plane distance using Image J software.2). The path of rays, sample, and detector are completely undervacuum . For the actual investigations,a 2-slit configuration and a beamstop with a 2 mm diameter was applied.SAXS/WAXSexperiments were executed by means of the multirange device Ganesha300 XL+ . We used Cu Kα radiation. Scattering intensities were accumulated by a2D-detector Pilatus 300 K  in three scattering ranges (with overlap), which weredistinguished by different distances sample-detector. The intensitieswere corrected by sample absorption. Bragg’s law is the basic d isthe lattice plane distance, θ is the half scattering angle, n is the order of reflection, and λ is the wavelength(Cu Kα radiation with 0.1542 nm). The total radial intensityprofile I(q) was calculated viaazimuthal averaging of the 2D frames and subsequent by merging ofthe partial intensity profiles; q is the  scattering vector.For samples  having isotropic scatteringbehavior in three-dimensional space (no any orientation effects),the measured intensity data near-zero diffraction angle (SAXS region)have to be corrected in general by a factor containing a sin(2θ)element or its squared form sin2(2θ) to reduce theintensities at small angles, which were generated from periodic structures(Gaussian or Lorentzian peaks with a peak maxima in correlation tothe Bragg periodicity) as well as dispersed particle systems (Gaussianpeaks around zero point). Applying Lorentz correction is helpful toenhance the visibility of the structures with periodicities , but it degrades the feature of particlescattering.37 Total radial intensity profiles were presented afterLorentz correction using the simplified The experiments were realized in asymmetric transmission. The bulksample was mounted free-standing, whereas the powder sample was enwrappedby thin aluminum foil. The primary data (intensity I) were recordedas 2D-scattering frames IL(lg q) and/orlg IL(lg d).as lg Figure S1, the FTIR spectra of epoxy(EPO) and its composite (EPO6Si_1.25) arereported together with the uncured resin (EPO_uncured) for comparison. The band at 914 cm–1 presentin the FTIR of uncured resin, which is attributed to the epoxy ring,almost disappears in the cured samples, implying the almost completeepoxy cure.38 Moreover, the increase, althoughsmall, of the band at 1070 cm–1 in the compositespectrum agrees with the presence of silica. The composite sample(in both powder and bulk form) was examined by X-ray scattering inthe WAXS, SAXS, and extremely small-angle region. The results arereported in Figure S2 (linear scattering curves: I = I(2θ)). The following observationscan be summarized:WAXS showsonly amorphous scattering with one broadscattering maximum at ∼18° in 2θ and one weak scatteringmaximum at ∼43°-44° caused by the epoxy matrix.SAXS shows two further maxima (∼5.8°and∼1.25°), that could be caused by unknown phase separationbetween the epoxy matrix and dispersed nanoparticles and/or the amorphousscattering from a special compound (organic/inorganic hybrid).ESAXS at very small scattering angles showsthe typicalpattern of well-distributed nanoparticles. This particle scatteringis caused by the silica particles in the bulk and silica alone . Thus, the intensity of particle scattering inthe powder sample must be quite high compared to the bulk. The slope– 2 respective 2 in the log–log curves 1a,b is tIn Figure S3 show that,at higher magnification, the nanoparticles appear to be consistingof equally spaced sheets, similar to the nanocrystalline structuresobserved through HRTEM by other researchers.39 The sheet thickness determined by a method described in the literature39 shown in Figure S3 is 0.34 nm, which is quite close to the lower value given by SAXSinvestigation  are expected to be formed through reaction of the APTESamino group and DGEBA oxirane ring  are reported.The hybrid shows an increase of both glass transition temperature(from 80 ± 1 to 84 ± 1 °C) and E’modulus mean values (from 2.7 ± 0.2 to 3.2 ± 0.2 ·109 Pa) corresponding to an increase of 19%. The increasesmay be ascribed to the strong interaction between the silica nanoparticlesand epoxy matrix through the hybrid molecules formed in the firststep and located at the epoxy/silica interface. As it is known,51 the hybrids are classified on the basisof the type of bonds at the O/I interface as “class I”in the case of weak bonding and “class II” in the caseof strong bonding. The DMA results suggest classifying the presenthybrid as “class II”.In particular, they might stabilize very tinywater/alcohol nanodropletsinside the epoxy resin that would play the role of the continuousphase of the emulsion. In these droplets, the typical sol–gelhydrolysis and polycondensation reactions might take place with theformation of very tiny silica particles see 1, step 2In the following, a moredetailed hypothesis concerning formationof the nuclei is developed. First of all the sheet structure of the“multi-sheet silica based nanoparticles “suggests thatthe hybrid APTS/epoxy molecule takes part to their formation. In fact,if formed only from TEOS, silica particles would be expected to form.Therefore, the nanoparticles are expected to have a hybrid organicinorganic (O/I) nature. It is also worth pointing out that, as a consequence,the nanoparticles may also be expected to possess a high affinityfor the epoxy matrix (we could say they are “epoxophilic”)owing to the presence, also at the surface, of the hybrid APTS/epoxymolecules, exposing epoxy groups externally. This is, otherwise, ingood agreement with the observed and above discussed change of glasstransformation temperature. We must expect that, owing to its nonpolarnature, TEOS dissolves in epoxy (where water cannot) and hydrolyzeswhen entering the micelles. Of course, also the silane head of thehybrid APTS/epoxy molecule constituting the micelle should be fullyhydrolyzed. If aggregation of micelles is admitted, a simple explanationis found to the involvement of both TEOS compared to the hybrid APTS/epoxymolecule in the formation of nuclei. It is useful reminding, in fact,that the specific surface of a droplet decreases when its radius increases.Therefore, the required number of hybrid molecules at the dropletborder is expected to decrease when the micelles do aggregate. Thehybrid molecules subtracted to the surface are available for the nucleiformation. Finally, the formation of the “epoxophilic”hybrid O/I nanoparticles may well cause separation of the micelleaggregate into a nanoparticle (the nuclei of the new phase) and oneor more new micelles containing the water residual from the hydrolysisand condensation reactions of alkoxy groups (of TEOS or APTS).A rough estimate proves that the number of hybrid APTS/epoxy moleculesavailable when micelle aggregation does occur is significant. It iswell known that the specific surface of a sphere is S/V = 3/R or S/m= 3/ρR . As a consequence, if, uponaggregation, the micelle diameter doubles, the specific surface ofthe “aggregated micelle” would be 2 times lower thanthe micelle one. This means that the mass of solution contained inthem would occupy a volume having a surface 2 times lower. So also,should decrease the need of hybrid APTS/epoxy molecules present atthe micelle surface. Therefore, a significant percentage (50%) ofthe hybrid APTS/epoxy molecules initially involved in the formationof micelle would be available for the nuclei formation.26 It was found:(a) the 29Si NMR spectra of studied hybrid material showedthat most of silicon atoms appeared to have been fully involved inthe condensation reactions; (b) the 13C CPMAS NMR spectrashowed that the intense ethoxy peaks were not detected in the mostshielded alkyl-C region, suggesting an approximately complete hydrolysisof ethoxy groups in both APTES and TEOS reagents. This is quite surprisingtaking into account31 that usually a significant residualorganic fraction does remain from the synthesis owing to incompletehydrolysis and polycondensation reaction  could be caused by a phase separationbetween epoxy matrix and dispersed nanoparticles (around 6 nm in size)so as already discussed. The second one (at ∼5.8°) couldwell be evoked by amorphous scattering from a special compound (organic/inorganichybrid) predicting for the residual micelles a diameter of about 1.50nm. The mechanism allows to give explanation also to NMR results reported,recently, for a composite of very close composition, prepared withexactly the same procedure.53 However, in the new hypothesized mechanism, the “solvent”and “surfactant” are not the common ones but, respectively,an epoxy resin at relatively high temperature (80 °C) and hybridmolecule obtained through the reaction of the “coupling agent″with the epoxy monomer. In the present case, silica-based nanoparticlesare formed “in situ” in DGEBA startingfrom simple precursor (TEOS and APTS) molecules both of which, tobe hydrolyzed, need water that is not soluble in DGEBA. The proposedmechanism allows to overcome the problem through the formation ofthe micelles making available the proper environment where the “smallstructural units” (required for the formation of nuclei ofthe silica based nanoparticles and their growth) may form. It is worthreminding that something similar was hypothesized54 to occur in the mechanism of formation of mesoporous silicananoparticles with radial wrinkle structure, which are typically formedin ternary systems of polar solvents (represented by water), nonpolarsolvents (oil), and surfactants. The reaction mixtures in those casescorrespond54 to the Winsor III system.It was hypothesized54 that TEOS dissolvedin the oil layer comes into contact with the water at the emulsioninterface where hydrolysis and condensation reactions occur. The mechanismallows us to explain, also, the peculiarities of recently publishedNMR results. Finally, the mechanism involves concepts borrowed fromthe nucleation/growth mechanism proposed initially by Tammann andthe Ostwald ripening process.Finally, HRTEM and SAXSallow us to have an insight into the nanoparticles(few nanometers in size), evidencing a multisheet structure that wouldform as the result of agglomeration and aggregation of micelle-likenanodroplets generated thanks to the hybrid molecules resulting fromthe reaction of epoxy with the coupling agent . The micelleswould assure the proper environment (presence of water which is notsoluble in the resin) where the “small structural units” may form. Nuclei would form on aggregation of micelles; crystalswould grow thanks to aggregation with micelles. The mechanism mayexplain the experimental results, in particular formation of particleswith a multisheet instead of a gel structure. It is, otherwise, worthmentioning that the relevant effects of the presence of “surfactants”on the sol–gel chemistry were recently recognized: new mesoporousgel textures were obtained when sol–gel network architectureswere controlled with the aid of the polymeric chain surfactant molecules.


Primary ciliary defects cause a group of developmental conditions known as ciliopathies. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitises cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. We identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalised, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. We demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1). MKS1 gene cause about 15% of MKS, a lethal neurodevelopmental condition that is the most severe ciliopathy  domain of the synaptotagmin-like and phospholipase families . MKS1 inM67 gene , and twoM67 gene . B9D1, BM67 gene  forming M67 gene . TZ protM67 gene . The comM67 gene , and mutM67 gene . For exaelopment . Previoud kidney .Other studies have shown that the ciliary apparatus restricts the activity of canonical Wnt/β-catenin signalling , althougHere, we describe the interaction and functional association of MKS1 with ciliary UPS components, specifically the E2 ubiquitin-conjugating enzyme UBE2E1  and the E3 ubiquitin ligating enzyme RNF34. In addition to ciliogenesis defects, loss of MKS1 causes deregulation of both proteasome activity and canonical Wnt/β-catenin signalling. These cellular phenotypes are also observed after loss of UBE2E1. MKS1 and UBE2E1 colocalise during conditions of cilia resorption, and levels of MKS1 and UBE2E1 are co-dependent. We show that in the absence of MKS1, levels of ubiquitinated proteins, including β-catenin, are increased. Furthermore, polyubiquitination of MKS1 is dependent on both UBE2E1 and RNF34, and lysine (Lys)63-linked polyubiquitination of MKS1 is dependent on UBE2E1. This suggests that regulation of intracellular signalling, specifically canonical Wnt/β-catenin signalling, can be regulated and constrained at the primary cilium by a ciliary-specific E2 and MKS1, a substrate-adaptor.-/-Mks1 mutant mice  also had de-regulated proteasome enzymatic activity , and significantly reduced both numbers of ciliated cells and mean cilium length , particularly under conditions of ciliary resorption  negative control cells . Furtherto Wnt3a . This wahibition , also coMKS1-mutated fibroblasts . The highest levels of MKS1 were observed in cells undergoing cilia resorption, when MKS1 and UBE2E1 co-localisation is the strongest. Despite modest statistical significance, this finding was consistent between biological replicates. Furthermore, expression of exogenous UBE2E1 led to moderate decreases in MKS1 levels for both shScr and shUbe2e1 knock-down cells, suggesting that UBE2E1 inhibits both MKS1 levels and polyubiquitination of this protein.UBE2E1 is an E2 ubiquitin conjugating enzyme, and we next tested the obvious hypothesis that it participates in polyubiquitination and targeting MKS1 for degradation. We therefore investigated if MKS1 is indeed tagged with ubiquitin chains and if absence of UBE2E1 affects ubiquitination of MKS1. We determined MKS1 levels and its polyubiquitination status in different ciliogenesis conditions, namely: proliferating cells ; ciliated cells (quiescent cells grown in serum-deprived medium); and cells undergoing ciliary resorption . shUbe2e1 cells had increased levels of polyubiquitinated cmyc-tagged MKS1 using TUBE (Tandem Ubiquitin Entity) assays. Total polyubiquitinated proteins from cell extracts were pulled-down using TUBEs bound to agarose beads, resolved by SDS-PAGE and analysed by western blotting using an anti-cmyc antibody. TUBE assays confirmed that MKS1 was polyubiquitinated , was shown to interact with Rpgrip1l  Research Ethics Committee (REC no. 08 /H1306/85) on 4th July 2008.Responsibility in the Use of Animals for Medical Research (July 1993) in accordance with UK Home Office regulations under the Project Licence no. PPL40/3349. B6;129P2-Mks1tm1a(EUCOMM)Wtsi heterozygous knock-out mice were derived from a line generated by the Wellcome Trust Sanger Institute and made available from MRC Harwell through the European Mutant Mouse Archive http://www.emmanet.org/ (strain number EM:05429). The G76V-GFPUb line (25) B6.Cg-Tg(G76V/GFPACTB-Ub*)1Dant/J (strain number 008111) was obtained from the Jackson Laboratory, Maine, USA. Genotyping was done by multiplex PCR on DNA extracted from tail tips or the yolk sac of E11.5-E15.5 embryos, or ear biopsies of adult mice. Primer sequences: exon 2 F: TGGGGAAGGACCTCATAGACT, exon 4 R: CGCCAGAATTCTCCAGTTTC, exon 4 F: AGCGTGGTTGTTCTTGATGA, exon 6 R: GGATTCCGCACTGAGACAAC, exon 16 F: AACCGGCGAATCTTCACTTA, exon 18 R: GGGGCTCACAAGGTCCTG. Proteasome inhibition treatment of Mks1 x G76V-GFPUb mice using MG-262 was carried out as previously described  cells were cultured in DMEM with 10% foetal calf serum at 37 °C/5% CO2. Cell-lines were maintained by weekly passaging under standard conditions and tested every 3 months for mycoplasma. Cell lines were sourced from American Type Culture Collection (ATCC) and used between passages 15–25. Cell-lines have been previously verified using arrayCGH and RNA-sequencing  with a compound heterozygous MKS1 mutation, and a female patient with a homozygous ASPM mutation, were immortalised following transduction with an amphotropic retrovirus encoding the hTERT catalytic subunit of human telomerase, and maintained in Fibroblast Growth Medium  supplemented with 0.2 mg/ml geneticin. Patient MKS-562, a compound heterozygote for the MKS1 mutations [c.472C > T]+[IVS15-7_35del29] causing the predicted nonsense and splice-site mutations [p.R158*]+[p.P470fs*562], has been described previously (Mouse inner medullary collecting duct (mIMCD3), human retinal pigment epithelium cells immortalised with human telomerase reverse transcriptase (hTERT-RPE1) and immortalised adult retinal pigment epithelium (ARPE19) cells were grown in Dulbecco’s minimum essential medium (DMEM)/Ham’s F12 supplemented with 10% foetal calf serum at 37 °C/5% COquencing  (Short Rquencing . MEFs weeviously . ProteasMKS1 was cloned into the pCMV-cmyc vector as described previously (Ube2e1 shRNA (Origene) were selected for using 0.5 μg/ml puromycin for five passages. Transfection with Dharmacon ON-TARGET PLUS siRNAs was carried out using Lipofectamine RNAiMAX according to the manufacturer’s instructions and as described previously , rabbit anti-GFP (‘Living Colors A.v. Peptide Antibody’) and mouse anti-UBE2E1 (BD Biosciences Inc); rabbit-anti-γ-tubulin and mouse anti-β-actin clone AC-15 (Abcam Ltd.); mouse anti-cyclin D1 clone A-12 (Santa Cruz Biotechnology Inc); rabbit anti-phospho-β-catenin and rabbit anti-β-catenin ; and mouse anti-mono- and polyubiquitinylated conjugates clone FK2 and rabbit anti-20S proteasome α7 subunit (Enzo Life Sciences Inc). Rabbit anti-MKS1 has been described previously . Seconda5 cells/well on glass coverslips in six-well plates, 24 hr before transfection and 48–96 hr before fixation. Cells were fixed in ice-cold methanol (5 min at 4 °C) or 2% paraformaldehyde (20 min at room temperature). Permeabilisation, blocking methods and immunofluorescence staining were essentially as described previously . We obtained Wnt3a- or Wnt5a-conditioned media from stably-transfected L cells with Wnt3a or Wnt5a expression vectors (ATCC). Control media was from untransfected L cells. Activities from firefly and Renilla luciferases were assayed with the Dual-Luciferase Reporter Assay system (Promega Corp.) on a Mithras LB940 (Berthold Technologies Inc) fluorimeter. Minimal responses were noted with co-expression of the FOP Flash negative control reporter construct. Raw readings were normalised with Renilla luciferase values. Results reported are from at least four independent biological replicates.For luciferase assays of canonical Wnt activity, we grew cells in 12-well plates and co-transfected with 0.5 μg SUPER-TOPFlash firefly luciferase construct  (or FOPFUBE2E1-FLAGcmyc was transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher Scientific inc) Cells were incubated with transfection complexes for 3 hr, and changed to normal growing medium for further 16 hr incubation. Cells were then incubated with 10 µM MG-132 for 5 hr and whole cell extracts (WCE) prepared as described previously . ProteinTo assess in vitro ubiquitination, we used a ubiquitination kit  according to the manufacturer’s protocol, supplemented with MKS1-HIS , UBE2E1-HIS , RNF34-HIS  and β-catenin-GST  fusion proteins in a total volume of 30 µl. Samples were incubated for 1.5 hr at 37 °C followed by SDS-PAGE and western blotting.Agarose-bound TUBE assays were used as recommended by the manufacturer . mIMCD3 cells were transiently transfected with cmyc-MKS1 and treated with proteasome inhibitor (MG-132 at 10 µM) for 2 hr before harvesting. Lysis buffer was based on RIPA supplemented with 50 mM Tris-HCl pH7.5, 0.15 M NaCl, 1 mM EDTA, 1% NP40, 10% glycerol, DUB inhibitors  and protease inhibitors. 5 µM BRISC was used as K63 deubiquitinating enzyme. In short, cells were harvested after incubation with proteasome inhibitor and proteins were extracted using TUBE lysis buffer following standard procedures. Protein concentration was measured using Lowry assay and about 750 µg of protein was used in the pull down. Cell lysates were incubated with uncoupled agarose beads to remove unspecific binding proteins and were subsequently incubated with equilibrated 40 µl TUBE-agarose beads for 2 hr at 4 °C on a rocker. Beads were spun down, washed, eluted and neutralised. Samples were then split into three for incubation with TBST, DUB and BRISC for 1 hr at 37 °C. Samples were run on SDS-PAGE followed by western blotting using standard protocols. Membranes were blotted with mouse anti-cmyc , Ub-HRP  and rabbit anti-β-catenin .Whole cell extracts (WCE) were prepared and co-IP performed essentially as described previously . Co-IPs Soluble protein was analysed by SDS-PAGE using 4–12% Bis-Tris acrylamide gradient gels and western blotting was performed according to standard protocols using either rabbit polyclonal antisera  or MAbs (x1000-5000). Appropriate HRP-conjugated secondary antibodies (Dako Inc) were used  for detection by the enhanced chemiluminescence ‘Femto West’ western blotting detection system (Pierce Inc). Chemiluminescence was detected using a BioRad ChemiDoc MP Imaging System and Image Lab software. Volumetric analysis of immunoblot bands was performed using Image Lab software (Bio Rad). Full blots are shown in the source data files associated with each figure and figure supplement, as appropriate.2 tests or other tests as detailed in figure legends as appropriate using InStat (GraphPad Software). Results reported are from at least three independent biological replicates.Normal distribution of data  was confirmed using the Kolmogorov-Smirnov test (GraphPad Software). Paired or unparied comparisons were analysed with either Student’s two-tailed t-test, χ It has long been recognized that ciliary dysfunction leads to increased canonical Wnt signaling but the mechanism has been elusive. Your work connecting β-catenin stability to Mks1 through Ube2e1 is an important advance in understanding this mechanism. I am certain that your work will stimulate more effort in this important area. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Gregory J Pazour as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Piali Sengupta as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Christoph Gerhardt (Reviewer #2).Thank you for submitting your article "Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript from Szymanska et al., explored MKS1 regulation of canonical Wnt signaling and uncovered a role for the E2 ubiquitin conjugating enzyme Ube2e1 in the degradation of phosphorylated β catenin. This paper addresses an important question in the field as to why ciliary dysfunction causes increased canonical Wnt signaling. The data convincing shows that the loss of MKS1 elevates canonical Wnt signaling. They then identify the E2 ligase Ube2e1 as an Mks1 binding protein by yeast two-hybrid and explore the role of Ube2e1 and Mks1 in regulating β catenin levels. The supporting data has significant problems that will need to be addressed before publication. First, many of the conclusions depend on small differences in band intensity. These differences are often not quantitated or if they are quantitated, it is not clear that the experiments were performed more than once. Statistical analysis also needs to be improved. Many experiments lack statistical tests; others use t-tests to compare multiple variables without any correction for multiple analysis. Secondly, as described below, the quality of several experiments needs to be improved if they are to be used to support the conclusions of the paper.Essential revisions:1. Data analysis is lacking. A number of figures lack quantitative analysis. Most lack details about the statistical analysis performed. It is not clear what "#" means in the graphs. It appears that a simple t-test were used for multiple comparisons without correction. Figure legends need to indicate the n of the experiment and the statistical test used.2. Why would increased proteasomal activity  cause GFP to accumulate in the UbG76V line? It seems that increased proteasomal activity would decrease (or not change) GFP accumulation.3. GST pulldown in Figure 2D is not convincing. The background in the control lane is darker than the experimental lane and could obscure a band of the intensity seen in the GST-UBE2E1 pulldown.4. Figure 3b: There is little or no difference between the "no colocalization" and "colocalization" images. There may be a quantitative difference in the centrosomal staining seen in the panels in 3a but this would need to be measured.5. Figure 5c. Western blots need to be added. Why is the level of nuclear Ube2e1 relevant to the work?6. The conclusion that increased expression of UBE2E1 decreases MKS1 levels (5d) is not convincing.7. The details of what was done in Figure 6b are lacking but a number of points are confusing. Why doesn't the band marked "MKS1cmyc-Ub"show up in the Ub western blot? Would we expect all ubiquitinated proteins to be detected in the Ub blot? If so, then this blot is not evidence of K63 polyubiquitination of the MKS1. The differences between the lanes in the c-myc blot are not convincing. I would expected the DUBs to reduce the extent of polyubiquitination, which should have brought the smears of poly-Ub-MKS1cmyc down the gel, but this is not seen.8. Figure 6c is problematic. First, both MKS1-GST and Ub-GST are used and a GST western blot is used as evidence of Ub ligation onto MKS1. Ub needs a different tag from its substrate to be convincing. Second, the patterns of the proteins in the blots do not match the guide at the top. Rnf34-His is found in lanes where it was not added. UbE2E1-Flag is only seen in the one of the lanes where it is supposed to be present. This seems to be an unduly complicated figure with UbE2E1-Flag and UbE2E1-His showing different results. There is no explanation of this in the figure legend or text.9. Figure 6e is too low quality to be interpreted.10. The staining of the neuroepithelium (S2) with the anti-acetylated tubulin antibody shows decreased cytoplasmic staining and no evidence of ciliary label. Better cilia markers, higher magnification and quantification are needed to make this point. This is also a problem in S1D where the MKS mutant cells have strong cytoplasmic label. It is unclear what the arrow is pointing at, but there is no evidence that it is a cilium.11. It was published that phospho-β-catenin (S33/S37/T41) is degraded by the proteasome at the base of cilia . Szymanska et al., showed that the amount of phospho-β-catenin (S33/S37/T41) is increased by MKS1 deficiency, although proteasome activity is enhanced in MKS1 mutant cells. How can this discrepancy be explained? The authors did not provide a sufficient explanation. An explanation could be that MKS1 affects Wnt signaling and proteasome activity in two different ways. But this needs to be shown. As they mentioned in the discussion, another transition zone protein, RPGRIP1L, positively regulates proteasome activity . Previously, it was shown that the loss of RPGRIP1L does not alter the amount of MKS1 at the ciliary transition zone of mouse embryonic fibroblasts  but maybe MKS1 deficiency results in an increased amount of RPGRIP1L at the transition zone of human and mouse fibroblasts leading to the higher proteasome activity the authors observed. Or mutations in MKS1 change the ciliary amount of BBS4 and/or OFD1 which also govern proteasome activity .12. Furthermore, some examinations should be performed in another way to allow a more precise interpretation. For example, the authors quantified phospho-β-catenin (S33/S37/T41) and β-catenin. In MKS1 deficient cells, the amount of both proteins is higher. In my opinion, an important control measurement is missing. The authors should additionally measure the amount of non-phospho-β-catenin (S33/S37/T41). Since Szymanska et al., suggest that MKS1 and UBE2E1 colocalize at the ciliary base, they should also measure the amount of hosphor-β-catenin (S33/S37/T41) not only by Western blotting  but also at the ciliary base by fluorescence intensity measurements in MKS1 deficient human or mouse fibroblasts. This kind of measurement was performed in hTERT-RPE1 cells which were treated with siRNA against MKS1  but in these cells the amount of γ-tubulin seems to be increased. Maybe, the increased amount of hosphor-β-catenin (S33/S37/T41) is caused by the higher amount of γ-tubulin. It cannot be excluded that several cilia overlap because this image lacks ciliary axoneme staining. In regard to the colocalization of MKS1 and UBE2E1, the authors should perform an analysis such as FRET or in situ PLA which confirms that both proteins localize close enough together to interact at the ciliary base.13. The proteasome activity assay uses the 20S fluorophore substrate Suc-LLVY-AMC to measure activity. The general view is that the proteasome degrades this substrate independently of ubiquitination . Despite of this, Szymanska et al., observe an enhanced proteasome activity in cells deficient for UBE2E1, an E2 ubiquitin-conjugation enzyme. This data should be carefully controlled in order to test whether the proteasome activity assay results are reliable. It would be helpful to check whether proteasome activity assays in which other substrates are used that are degraded by the proteasome without ubiquitination provide the same results. If this is the case, UBE2E1 may indirectly regulate proteasome activity by taking part in the ubiquitination of a protein that regulates proteasome activity.14. In human there are approximately 40 E2 enzymes, therefore each E2 is likely to act with multiple E3s (there are over a 1000) with E3s likely to be the main driver of target specificity. Authors have not controlled their experiments, or at least have not showed it in the manuscript, with any other E2s. Consequently, there is a real risk that the findings simply confirm that cilia and MKS1 are regulated by UPS rather than that UBE2E1 is a specific regulator in this system.15. With regard to immunoprecipitations and ubiquitination assays. Firstly inputs  and/or loading controls are not always shown  which makes interpretation of results difficult.16. Details are lacking about the conditions of the IP experiments used to detect ubiquitinated proteins . If these were performed under native conditions, the ubiquitin being detected by western blot could be on any protein that complexes with the target of the precipitation. E2 ligases may have a large number of interactors.17. As far as the UBE2E1 knockdown model. In the MS analysis in Figure 3, I am quite surprised that there is still such a strong recovery of UBE2E1 peptides from UBE2E1 silenced line. Is this not an impediment to this model? Does this not suggest that this cell line still has sufficient, functioning UBE2E1? Also again, it is hard to estimate if the MKS1 specific observations would not be replicated by silencing other E2s.18. Lack of inputs in Figure 6a,b,d and e make interpretation of the critical claims from this manuscript difficult. If there is an effect of UBE2E1 on MKS1 total protein levels inputs of UBE2E1, MKS1 and loading controls for all samples in 6a 6b and 6d should clarify this. Currently, the only demonstration of the claimed protein increase is in Figure 5d and it is not repeated 3 times to allow for statistical analysis. Furthermore, it looks very marginal  and unlikely to be significant. It is possible that even if direct, changes caused by UBE2E1 to MKS1and vice versa are completely irrespective of protein levels or stability and affect localization and function through chains other than K48.19. Authors should clarify statements regarding degradation and UBE2E1 dual function, as alternative explanations have not been ruled out. UBE2E1 action on MKS1 could be indirect. The increase in b-catenin levels in Figure 6d is very hard to interpret without loading controls.20. There are multiple alternative explanations for the observations presented in this manuscript, which renders figure 7 a bit uncertain. For example, UBE2E1 could be involved in ubiquitination of an MKS1 targeting E3 or DUB and that could be the reason for the observed changes. It would be more forthcoming to highlight this in the manuscript.References:Corbit, K., Shyer, A., Dowdle, W., Gaulden, J., Singla, V., Chen, M., Chuang, P., Reiter, J., 2008. Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 70-76.Gan, J., Leestemaker, Y., Sapmaz, A., Ovaa, H., 2019. Highlighting the Proteasome: Using Fluorescence to Visualize Proteasome Activity and Distribution. Front. Mol. Biosci. 6, 14.Gerhardt, C., Lier, J., Burmühl, S., Struchtrup, A., Deutschmann, K., Vetter, M., Leu, T., Reeg, S., Grune, T., Rüther, U., 2015. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium. J. Cell Biol. 210, 115-133.Liu, Y., Tsai, I., Morleo, M., Oh, E., Leitch, C., Massa, F., Lee, B., Parker, D., Finley, D., Zaghloul, N., Franco, B., Katsanis, N., 2014. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators. J. Clin. Invest. 124, 2059-2070.Wheway, G., Abdelhamed, Z., Natarajan, S., Toomes, C., Inglehearn, C., Johnson, C., 2013. Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome.. Dev. Biol. 377, 55-66.Wiegering, A., Dildrop, R., Kalfhues, L., Spychala, A., Kuschel, S., Lier, J., Zobel, T., Dahmen, S., Leu, T., Struchtrup, A., Legendre, F., Vesque, C., Schneider-Maunoury, S., Saunier, S., Rüther, U., Gerhardt, C., 2018. Cell type-specific regulation of ciliary transition zone assembly in vertebrates. EMBO J. 37, pii: e97791. Essential revisions:1. Data analysis is lacking. A number of figures lack quantitative analysis. Most lack details about the statistical analysis performed.All information about statistical analysis is now included in figure legends and in the Methods and Materials section.It is not clear what "#" means in the graphs.The relevant graphs have been redrawn to make clear the pair-wise comparisons under testIt appears that a simple t-test were used for multiple comparisons without correction.Statistical tests corrected for multiple comparisons (ANOVA) have been used for Figure 3b and 3d and 6a.Figure legends need to indicate the n of the experiment and the statistical test used.We have addressed this point in all figure legends as appropriate2. Why would increased proteasomal activity  cause GFP to accumulate in the UbG76V line? It seems that increased proteasomal activity would decrease (or not change) GFP accumulation.UbG76V embryos were treated with a proteasome inhibitor that will prevent degradation of ubiquitinated proteins. Although this procedure was detailed in the Methods and Materials section, we now make this clear in the main text and figure legends, in order to aid interpretation of our observation that loss of Mks1 caused abnormal accumulation of ubiquitinated proteins. Since we also observed a concomitant increase in proteasome activity, we interpret this increase as a response to facilitate the degradation of abnormal amounts of polyubiquitinated proteins. Therefore, when the proteasome is inhibited we observe higher levels of GFP.3. GST pulldown in Figure 2D is not convincing. The background in the control lane is darker than the experimental lane and could obscure a band of the intensity seen in the GST-UBE2E1 pulldown.We display a new western blot in Figure 2d from a separate biological replicate of this pulldown4. Figure 3b: There is little or no difference between the "no colocalization" and "colocalization" images. There may be a quantitative difference in the centrosomal staining seen in the panels in 3a but this would need to be measured.We have increased the size of magnified insets in Figure 3b and 3d to make the punctate staining at basal bodies more clear. We have attempted to calculate co-localization values between the red (UBE2E1) and green (MKS1) channels (using z-stacks of these channels), but this has been difficult to compare between biological replicates for the two cell-lines because of the variable staining pattern of UBE2E1 in nuclei. We therefore feel that a simple scoring based on presence or absence of UBE2E1 at basal bodies is the most representative of the data.5. Figure 5c. Western blots need to be added. Why is the level of nuclear Ube2e1 relevant to the work?We have included the western blots in Figure 5c as requested. We have analysed the overall levels of UBE2E1 localization, including that localized at nuclei, in the “staining intensity” data because it is consistent with the overall increase of protein levels that we observe following western blotting of whole cell extracts. Both data support our contention that MKS1 and UBE2E1 protein levels are co-regulated.6. The conclusion that increased expression of UBE2E1 decreases MKS1 levels (5d) is not convincing.In Figure 5d, we have used a clearer western blot from a separate biological replicate of this experiment. . This experiment was conducted three times with consistent results that all demonstrate the co-dependency of protein levels between MKS1 and UBE2E1.7. The details of what was done in Figure 6b are lacking but a number of points are confusing. Why doesn't the band marked "MKS1cmyc-Ub"show up in the Ub western blot?The details of methodology for the assay shown in Figure 6b are now included in the Materials and Methods and in the figure legend. In response the second point, the pull-downs bring down all ubiquitinated proteins (the agarose beads are cross-linked to four separate ubiquitin-binding domains) which will be detected by the anti-ubiquitin antibody. Consequently, the specific “MKS1cmyc-Ub” band will be lost in the smear of all ubiquitinated proteins.Would we expect all ubiquitinated proteins to be detected in the Ub blot? If so, then this blot is not evidence of K63 polyubiquitination of the MKS1.We agree that all ubiquitinated proteins will be detected in the Ub blot, but this blot is included  as an essential control to show that the K63-specific de-ubiquitinating enzyme (DUB) is active and that the TUBE assay has worked. The top panel of Figure 6b with the anti-cmyc blot shows decreased levels of MKS1 after K63-DUB treatment in comparison to TBST (100 and 149 vs 80 and 121 AUs), suggesting that MKS1-cmyc is at least partially polyubiquitinated with K63 ubiquitin chains.The differences between the lanes in the c-myc blot are not convincing. I would expected the DUBs to reduce the extent of polyubiquitination, which should have brought the smears of poly-Ub-MKS1cmyc down the gel, but this is not seen.We now include normalised band intensities for polyUb-cmycMKS1 under the blots in the top panel of Figure 6b, which shows modest yet consistent decreases in polyubiquitination after DUB and K63DUB treatments, for multiple biological replicates of this experiment. However, we agree that de-ubiquitination was not efficient, even following the manufacturer’s protocol, meaning that quantitation between replicates has been problematic. We therefore provide data in Figure 6b as representative of this set of experiments.8. Figure 6c is problematic. First, both MKS1-GST and Ub-GST are used and a GST western blot is used as evidence of Ub ligation onto MKS1. Ub needs a different tag from its substrate to be convincing.We agree with this important point, and have gone to considerable efforts to repeat the entire experiment using untagged Ub and His-tagged MKS1 . This new data shows both mono-ubiquitination of MKS1 and poly-ubiquitination of β-catenin that is dependent on MKS1. .Second, the patterns of the proteins in the blots do not match the guide at the top. Rnf34-His is found in lanes where it was not added.We apologize for this error in labelling the lanes and have corrected the figure.UbE2E1-Flag is only seen in the one of the lanes where it is supposed to be present.We have replaced the input UBE2E1 with a His-tagged protein, and the UBE2E1-FLAG data is included in Figure 6—figure supplement 1a (see above).This seems to be an unduly complicated figure with UbE2E1-Flag and UbE2E1-His showing different results. There is no explanation of this in the figure legend or text.We have repeated and simplified the presentation of this entire set of assays . Figure 6c demonstrates that UBE2E1 undergoes auto-ubiquitination and that ubiquitination is enhanced but not dependent on RNF34. MKS1 inhibits UBE2E1 ubiquitination, suggesting that this could be the basis for the co-dependency of protein levels . This action is attenuated by addition of β-catenin, but β-catenin by itself does not inhibit UBE2E1 ubiquitination, suggesting that UBE2E1 and MKS1 are co-regulators of β-catenin ubiquitination .9. Figure 6e is too low quality to be interpreted.This experiment has been repeated and is now shown in Figure 7b. Consistently for all replicates, we observe an increase of polyubiquitinated β-catenin following loss of MKS1 and over-expression of active  UBE2E1. This suggests that when MKS1 and active UBE2E1 are present, normal poly-ubiquitination and degradation of β-catenin is taking place. Upon loss of MKS1, this process is disrupted leading to abnormally high levels of highly polyubiquitinated β-catenin. Poly-ubiquitination of β-catenin is much reduced in the presence of inactive  UBE2E1, again suggesting that UBE2E1 and MKS1 are co-regulators of β-catenin ubiquitination.10. The staining of the neuroepithelium (S2) with the anti-acetylated tubulin antibody shows decreased cytoplasmic staining and no evidence of ciliary label. Better cilia markers, higher magnification and quantification are needed to make this point. This is also a problem in S1D where the MKS mutant cells have strong cytoplasmic label. It is unclear what the arrow is pointing at, but there is no evidence that it is a cilium.Unfortunately, in our hands, the anti-acetylated tubulin antibody gives variable results on fresh-frozen tissue sections and primary dermal fibroblasts, so we have removed this data in Suppl. Figure 2. We have retained the data showing the increase of activated β-catenin, consistent with data shown in Figure 1a-b.This is also a problem in S1D where the MKS mutant cells have strong cytoplasmic label.We agree, but IF staining with this label does not preclude accurate quantitation of cilia incidence and length in these cells and this does seem to be a consistent cellular phenotype in MKS1-mutated cell-lines .11. It was published that phospho-β-catenin (S33/S37/T41) is degraded by the proteasome at the base of cilia . Szymanska et al., showed that the amount of phospho-β-catenin (S33/S37/T41) is increased by MKS1 deficiency, although proteasome activity is enhanced in MKS1 mutant cells. How can this discrepancy be explained? The authors did not provide a sufficient explanation.We now make the following potential mechanism clearer in the Discussion: phospho-β-catenin after being phosphorylated needs to be ubiquitinated, probably by UBE2E1  at the base of the cilium. Our data indicates that MKS1 is necessary to facilitate correct ubiquitination of β-catenin by UBE2E1. Due to the lack of MKS1, UBE2E1 levels increase, which increases polyubiquitination of phosphorylated β-catenin localized at the ciliary base. We observe a concomitant increase in proteasome activity, and suggest that this up-regulation is in response to increased levels of poly-ubiquitinated proteins, including β-catenin, but the levels appear to overload the ability of the proteasome to process them for degradation. As a consequence, there is abnormal accumulation of β-catenin at the ciliary base, where it awaits its degradation, leading to de-regulation of canonical Wnt signalling.An explanation could be that MKS1 affects Wnt signaling and proteasome activity in two different ways. But this needs to be shown. As they mentioned in the discussion, another transition zone protein, RPGRIP1L, positively regulates proteasome activity . Previously, it was shown that the loss of RPGRIP1L does not alter the amount of MKS1 at the ciliary transition zone of mouse embryonic fibroblasts  but maybe MKS1 deficiency results in an increased amount of RPGRIP1L at the transition zone of human and mouse fibroblasts leading to the higher proteasome activity the authors observed. Or mutations in MKS1 change the ciliary amount of BBS4 and/or OFD1 which also govern proteasome activity .We have addressed this point by using the best available commercial antibody, against RPGRIP1L, and doing a series of western blots following both MKS1 knockdown and in MKS1-mutated fibroblasts . RPGRIP1L protein levels were indeed increased in MKS1-mutated fibroblasts compared to normal controls (HDF), but this result was not confirmed by MKS1 knockdown. We therefore conclude that the potential regulatory action of MKS1 on RPGRIP1L warrants further investigation, but is somewhat beyond the current remit and focus of this study.12. Furthermore, some examinations should be performed in another way to allow a more precise interpretation. For example, the authors quantified phospho-β-catenin (S33/S37/T41) and β-catenin. In MKS1 deficient cells, the amount of both proteins is higher. In my opinion, an important control measurement is missing. The authors should additionally measure the amount of non-phospho-β-catenin (S33/S37/T41).We have measured levels of non-phospho-β-catenin in both MKS1 knockdown and in MKS1-mutated fibroblasts . Β-catenin protein levels were marginally increased following MKS1 knockdown but this result was not confirmed in MKS1-mutated fibroblasts. We conclude that there does not appear to be an overall effect on the levels of non-phospho-β-catenin.Since Szymanska et al., suggest that MKS1 and UBE2E1 colocalize at the ciliary base, they should also measure the amount of phospho-β-catenin (S33/S37/T41) not only by Western blotting  but also at the ciliary base by fluorescence intensity measurements in MKS1 deficient human or mouse fibroblasts.We present new data in Figure 7—figure supplement 1b to support the data in Figure 7c, showing an increase in phospho-β-catenin at the base cilia but no overall change in non-phospho-β-catenin levels following MKS1 knockdown. We used γ-tubulin staining to define regions-of-interest to ensure that the analyses were limited to the ciliary base; there is no perturbation of γ-tubulin levels following MKS1 knockdown .This kind of measurement was performed in hTERT-RPE1 cells which were treated with siRNA against MKS1  but in these cells the amount of γ-tubulin seems to be increased.We have confirmed that there is no difference in γ-tubulin levels at the ciliary base following MKS1 knockdown . We have changed the field of view presented in Figure 7c to clarify the observation.Maybe, the increased amount of phospho-β-catenin (S33/S37/T41) is caused by the higher amount of γ-tubulin. It cannot be excluded that several cilia overlap because this image lacks ciliary axoneme staining.MKS1 knock-down or mutant cells have multiple cilia .We agree that this is a possibility, but we have never observed that In regard to the colocalization of MKS1 and UBE2E1, the authors should perform an analysis such as FRET or in situ PLA which confirms that both proteins localize close enough together to interact at the ciliary base.We used multiple assays that included Y2H, GST pulldown and IP, and IF microscopy to show that MKS1 and UBE2E1 biochemically interact and co-localise. Importantly, Y2H assays indicate that there is a direct interaction between MKS1 and UBE2E1, and we therefore judge that proximity assays such as FRET and PLA would be merely confirmatory.13. The proteasome activity assay uses the 20S fluorophore substrate Suc-LLVY-AMC to measure activity. The general view is that the proteasome degrades this substrate independently of ubiquitination . Despite of this, Szymanska et al., observe an enhanced proteasome activity in cells deficient for UBE2E1, an E2 ubiquitin-conjugation enzyme. This data should be carefully controlled in order to test whether the proteasome activity assay results are reliable. It would be helpful to check whether proteasome activity assays in which other substrates are used that are degraded by the proteasome without ubiquitination provide the same results.In all relevant assays, we have used proteasome inhibitors to control for its activity. These experiments were conducted in three different cell lines  always with the control of proteasome inhibitor treatment. In addition, we have used “irrelevant control” cells , which have proteasome activity levels similar to those observed in control cells. We therefore judge that the experiments are well-controlled as presented and that they reflect a reproducible biological effect. Furthermore, in data that we present here, proteasome protein levels appear to be up-regulated, consistent with the increase in activity . We therefore do not think that additional proteasome activity assays will provide additional insight above the conventional, accepted assays of activity that we already report.If this is the case, UBE2E1 may indirectly regulate proteasome activity by taking part in the ubiquitination of a protein that regulates proteasome activity.We agree that this is a possibility, but another alternative is that loss of UBE2E1 causes dysregulation of another E2 or E2s that cause aberrant increase of polyubiquitinated proteins and hence increase in proteasome activity in degradation of polyubiquitinated proteins. Discriminating between these alternatives is, we feel, beyond the focus of the manuscript.14. In human there are approximately 40 E2 enzymes, therefore each E2 is likely to act with multiple E3s (there are over a 1000) with E3s likely to be the main driver of target specificity. Authors have not controlled their experiments, or at least have not showed it in the manuscript, with any other E2s. Consequently, there is a real risk that the findings simply confirm that cilia and MKS1 are regulated by UPS rather than that UBE2E1 is a specific regulator in this system.degradation of MKS1 (rather than co-regulation) is likely to be mediated by another E2  because polyubiquitination of MKS1 is inhibited, rather than enhanced, by UBE2E1 .The specific localization and interactions of UBE2E1, and reciprocal co-regulation of MKS1 and UBE2E1, argue that UBE2E1 is a specific co-regulator. In particular, the dominant negative (DN) mutant C131S of UBE2E1 has specific and significant effects on ciliogenesis. However, we agree that 15. With regard to immunoprecipitations and ubiquitination assays. Firstly inputs  and/or loading controls are not always shown  which makes interpretation of results difficult.Input material  and loading controls are now shown for the TUBE assays . Input material is also shown for the pulldowns in Figure 2d-f .16. Details are lacking about the conditions of the IP experiments used to detect ubiquitinated proteins . If these were performed under native conditions, the ubiquitin being detected by western blot could be on any protein that complexes with the target of the precipitation. E2 ligases may have a large number of interactors.We apologize for this oversight and now provide further details of the methodology used in our IP experiments. We now make clear that we used denaturing conditions for the western blots. We note that TUBE assays  pull-down all poly-ubiquitinated proteins and the western blot is then performed to visualize the protein of interest.17. As far as the UBE2E1 knockdown model. In the MS analysis in Figure 3, I am quite surprised that there is still such a strong recovery of UBE2E1 peptides from UBE2E1 silenced line. Is this not an impediment to this model? Does this not suggest that this cell line still has sufficient, functioning UBE2E1? Also again, it is hard to estimate if the MKS1 specific observations would not be replicated by silencing other E2s.Ube2e1 cell-lines . We do not observe recovery of Ube2e1 peptides under these conditions, but there are moderate levels of other E2s . Successful Ube2e1 knock-down in the shUbe2e1 cell-line is demonstrated in Figure 4c.We think that the reviewers may have misinterpreted what is shown in Figure 4e: the peptide count ratios are for the various different protein species immunoprecipitated by anti-MKS1 from the shScrambled control vs the sh18. Lack of inputs in Figure 6a,b,d and e make interpretation of the critical claims from this manuscript difficult. If there is an effect of UBE2E1 on MKS1 total protein levels inputs of UBE2E1, MKS1 and loading controls for all samples in 6a 6b and 6d should clarify this.Input material is now shown for Figures 2d-f, 6b-c and 7a-b . Input recombinant proteins are also shown individually for the in vitro ubiquitination assays .Currently, the only demonstration of the claimed protein increase is in Figure 5d and it is not repeated 3 times to allow for statistical analysis. Furthermore, it looks very marginal (numbers are not greatly affected either) 113-107-96…In Figure 5d, we have used a clearer western blot from a separate biological replicate of this experiment ...with appreciation that  and unlikely to be significant.As mentioned above (point 6), this experiment was conducted three times with consistent results that all demonstrate the co-dependency of protein levels between MKS1 and UBE2E1. We have not provided a statistical analysis of the replicates for this assay exactly for the reasons stated by the reviewer for this assay .It is possible that even if direct, changes caused by UBE2E1 to MKS1and vice versa are completely irrespective of protein levels or stability and affect localization and function through chains other than K48.The TUBE data shown in Figure 6b suggests that MKS1 is modified by K63 ubiquitination. We have also attempted to confirm this directly by LC-MS/MS analysis of peptide species, but unfortunately, in our hands, the B9 domain appears to be particularly resistant to standard trypsin digestion and even the trypsin/Lys-C protocol for tightly folded proteins does not provide coverage of the predicted modified lysine groups in MKS1.19. Authors should clarify statements regarding degradation and UBE2E1 dual function, as alternative explanations have not been ruled out. UBE2E1 action on MKS1 could be indirect.The interpretation of our data is that the direct interaction between MKS1 and UBE2E1 mediates K63 mono/bi-ubiquitination of MKS1, modulating MKS1 function, ciliogenesis and other known downstream functions such as β-catenin polyubiquitination. However, we assume that another E2 or E2s regulate degradation/polyubiquitination of MKS1.The increase in b-catenin levels in Figure 6d is very hard to interpret without loading controls.To aid interpretation of our data, we have provided loading controls and a new TUBE assay has been performed .20. There are multiple alternative explanations for the observations presented in this manuscript, which renders figure 7 a bit uncertain. For example, UBE2E1 could be involved in ubiquitination of an MKS1 targeting E3 or DUB and that could be the reason for the observed changes. It would be more forthcoming to highlight this in the manuscript.In response to point 19 above, we now provide alternative interpretations of our data in the Discussion, but the most parsimonious explanation of our findings are summarized in Figure 7d. in the main text we now state: “…alternative interpretations of our data are that UBE2E1 could occupy an E2 binding position on MKS1 preventing MKS1 degradation, or that MKS1 binds to UBE2E1 to facilitate UBE2E1 ubiquitination and degradation”


Anoectochilus roxburghii polysaccharide (ARP) or exercise (EX) exhibited the beneficial effects on metabolic health. However, the synergetic beneficial effects of ARP and EX as a combined intervention on obesity-induced metabolic disorders remain largely obscure. Male C57BL/6 mice were fed a high-fat diet (HFD) and intervened with ARP and EX for 12 continuous weeks. The results indicated that the ARP, EX, and ARP combined with EX treatment group regulated lipogenesis by suppressing the fatty acid pathway, dampening the system oxidative stress by stimulating Nrf2-mediated phase II enzyme system, and promoting the mitochondrial function by activating the mitochondrial complexes and PGC-1α in HFD mice. More importantly, the combination of ARP and EX showed an even greater beneficial effects relative to either ARP or EX alone, especially in decreasing reactive oxygen species (ROS) level and increasing adenosine triphosphate (ATP) content. Taken together, these findings further confirmed that ARP and EX could be effective interventions on obesity-induced metabolic abnormalities, and that the combination of ARP and EX exhibited the beneficial synergetic effects.Metabolic syndrome is a cluster of metabolic disorders that threatens public health. Nevertheless, its exact mechanism and relative intervention remain largely obscure. Accumulating evidence indicate that tither  The metabolic syndrome, characterized by obesity and its related metabolic diseases, has become a major public health concern in modern society . The patFurther, metabolic disorder has been proved to be strongly related to the system oxidative stress, and therefore affects metabolic state , 13. TheAnoectochilus roxburghii is a classical traditional Chinese herb and called “king's medicine” in southern China  has been attributed with antidiabetic effects in the improvement of glucose and lipid metabolism  intervention are still considered as clinically beneficial mainstays to promote metabolic health , 20. To rn China . Owing t effects –25. Sometabolism , 26, 27.The purpose of the present study was to investigate the synergetic effects of ARP combined with EX in C57BL/6 HFD-fed mice, and to further explore the detailed mechanism underlying the protective effects on obesity-induced metabolic disorders. The findings would unveil a better understanding of the combination intervention in improving metabolic health and find more potential therapy targets.Primary antibodies against NF-E2 related factor , heme oxygenase-1  and NAD(P)H/quinone oxidoreductase  were obtained from Santa Cruz Biotechnology . Antibodies against fatty acid synthase , acetyl-coenzyme A carboxylase 1 , peroxisome proliferator-activated receptor gamma coactivator-1α  and β-actin (#58169) were purchased from Cell Signaling Technology . Antibodies against NDUFS3 (#459130), SDHB (#459230), UQCRC1 (#459140), MTCO1 (#459600), and ATP5A1 (459240) were acquired from Thermo Fisher Scientific .A. roxburghii was purchased from the Drug Store Pharmacy of Fuzhou . To examine the optimum conditions for the extraction of A. roxburghii polysaccharose (ARP), the influence of various factors were tested, including ultrasonic temperature, power, time, and material liquid ratio. Briefly, dried A. roxburghii was exhaustively extracted in distilled water according to the liquid-to-material ratio of 1:3 (v/v), followed by an ultrasound at 200 W for 30 min, and then supplemented with distilled water with the ratio of 1:35 (v/v) at 80°C for 2 h. Next, the solution was centrifuged and concentrated with a rotary evaporator at 80°C. Then, the concentration was deproteinated with a Sevag method, washed with acetone and ether, and finally precipitated with EtOH for purification. The average yield of ARP is ~3.16% (w/w). For ARP analysis, the phenol-sulfuric acid method (with glucose as the standard) was performed to measure the degree of the purity of ARP , namely, normal-food diet , high-fat diet , HFD plus daily administration of 400 mg/kg/day ARP (HFD+ARP), HFD plus exercised , and HFD plus daily administration of ARP plus exercised (HFD+ARP+EX). All the administrations were conducted between 9 and 10 o'clock in the morning, and body weight was measured every week. All animal experiments were approved by the Animal Ethics Committee in Fujian University of Traditional Chinese Medicine and performed according to the guidelines of animal care and use for scientific purposes.Male C57BL/6 mice  in this study were purchased from the Shanghai Laboratory Animals Center (SLAC). Mice were kept under a condition of 24 ± 0.5°C, relative humidity of 60% with a 12 h light/dark cycle. After 1 week of adaptation, the mice have been randomly divided to five groups  in the mice. Blood was taken from the tail vein every 30 min for over 2 h, and the concentration of plasma glucose was finally examined by an Accu-Chek meter .After mice were sacrificed, small portions of liver tissues were quickly excised, weighed, homogenized in chilled PBS, and the supernatant was separated by centrifugation . Meanwhile, blood samples were collected by cardiac puncture, and the serum was obtained with centrifugation . The tissue and serum samples were prepared for the following experiment.The concentrations of total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPX), reduced glutathione (GSH), glutathione S-transferase (GST) and glucose in serum or liver homogenates were examined by biochemical kits . The content of insulin, tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin-6 (IL-6) was analyzed by enzyme-linked immunoassay (ELISA) kits (Nanjing Jiancheng). The content of ATP was examined with bioluminescent kits .Liver tissues were removed, rinsed with chilled phosphate-buffered saline (PBS), then fixed in 10% paraformaldehyde solution. The treated-tissues have been embedded in paraffin, sectioned (thickness 4–5 μm), stained with hematoxylin and eosin (H&E), and visualized by Olympus BX71 microscope.The level of protein carbonyls was examined by the Oxyblot protein oxidative detection kit . The content of protein carbonyls was determined using western blot assay, and the sample amount was measured using Coomassie brilliant blue.The level of intracellular ROS has been measured using a fluorescence with 2'7'-dichlorofluorescein (DCF-DA). The fluorescence wavelength with 488 nm excitation and 535 nm emission was used for detection by a microplate fluorometer.The protein sample was extracted using immunoprecipitation (IP) lysis buffer, and then concentrated by the bicinchoninic assay (BCA) kit . The equal amounts of samples were injected in an 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and further transferred onto a hydrophilic polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat-milk and then incubated with the appropriate antibodies. In another day, the membrane was incubated with the secondary antibodies. Finally, the protein band was visualized by an enhanced chemiluminescence (ECL) plus reagent.p < 0.05 was utilized as statistically significant.All the data in the study were exhibited as mean ± standard error of the mean (S.E.M). The one-way ANOVA was analyzed by GraphPad Prism 8.0  for statistics. For analysis, The high-fat diet C57BL/6 mouse is a universal used animal model for the study of obesity-associated diabetes. Being overweight or obese is believed to accelerate the risk of developing MetS . In the At the 12th week, an OGTT was detected and revealed that the insulin sensitivity showed an obvious decrease in the HFD group relative to the normal control group, which was increased in the ARP and EX treatment group . The serThe HFD+ARP, HFD+EX, and HFD+ARP+EX showed a 53.2, 28.4, and 62.9% decrease as compared to the HFD group, suggesting potential insulin resistance . More imInflammation has emerged as a critical factor in the main cause of T2DM complications as it may trigger the onset and progression of diabetes . As expeHyperlipidemia is usually manifested as abnormal lipid metabolism . The HFDOxidative stress, an imbalance between oxidants and antioxidants, has been considered as a critical contributor in the pathogenesis of obesity-associated disease . RelativMitochondria are the cytoplasmic organelles strongly associated with cellular energy metabolism . To explDue to unhealthy dietary lifestyle, the prevalence of MetS has reached global epidemic proportions. As a critical risk factor of MetS, obesity is one of the most common lipid abnormalities and has been proved to be strongly associated with metabolic-related disorders, including diabetes, cardiovascular and fat liver diseases –40. The It is well-established that MetS is accompanied by an increased risk of hyperlipidemia, and the excessive fat or lipid significantly deposited under this state . ExcessiPrevious studies have revealed that the MetS disorder is closely associated with excessive lipid accumulation. In the study, it was discovered that the liver contents of TG and TC in HFD-fed mice exhibited a significantly elevated relative to the normal control group. The HFD+ARP, HFD+EX, and HFD+ARP+EX group sufficiently decreased the TG content, however, only the HFD+ARP+EX group could alleviate the abnormality in the TC content. In addition, the combination of ARP and EX exhibited an even greater diminishing relative than either ARP or EX alone, which was further confirmed by histology staining in the livers. As a major contributing factor to lipid biosynthesis, the genes involved in the fatty acid synthesis (FAS and ACC1) exhibited a significant higher expression in obese mice than those in normal control mice, leading to lipid accumulation , 48. In Apart from the upregulation of lipogenesis, the pathogenesis of MetS is usually characterized by chronic inflammation and oxidative stress , 50. PreMitochondria exerts crucial roles on energy expenditure and ROS production. Their dysfunction affects not only fat homeostasis but also ROS and cytokines levels, which further induces oxidative stress and chronic inflammation and contributes to the progression of MetS , 55. TheIn conclusion, the current research demonstrated that combined ARP with EX intervention could protect livers in response to obesity-induced metabolic disorders by upregulating the PGC-1α-mediated mitochondrial biogenesis. Compared to either ARP or EX treatment, this combination exhibited greater effective synergetic benefits through reducing lipid accumulation, dampening inflammation, oxidative stress, and improving mitochondrial function in the livers. Together, these findings provide an additional evidence for the use of a combination of diet and exercise might be a feasible non-pharmacological therapy intervention for ameliorating obesity-induced metabolic disorders.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Animal Ethics Committee in Fujian University of Traditional Chinese Medicine and performed according to the guidelines of animal care and use for scientific purposes.CC and XH: project administration. CC, MK, and QW: data curation. QX, WL, MY, and SL: investigation. CC, QW, QX, and WL: data analysis. CC, XH, and JT: writing. All authors have read and agreed to the published version of the manuscript.This study was supported by grants from the Scientific Research Foundation for the High-level Talents, Fujian University of Traditional Chinese Medicine .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.