The formation of fluorite (CGO) and perovskite(LCM) phases in the nanocomposite electrode is confirmed by differentstructural and microstructural techniques. The intimate mixture ofLCM and CGO phases inhibits the grain growth, retaining the nanoscalemicrostructure even after annealing at 1000 °C with a grain sizelower than 50 nm for LCM-CGO compared to 200 nm for pure LCM. Thesynergetic effect of nanosized LCM and CGO by combining their highelectronic and ionic conductivity, respectively, leads to efficientand durable symmetrical electrodes. The best electrochemical propertiesare found for 50 wt % LCM-CGO, showing polarization resistance valuesof 0.29 and 0.09 Ω cm2 at 750 °C in air andH2, respectively, compared to 2.05 and 1.9 Ω cm2 for a screen-printed electrode with the same composition.This outstanding performance is mainly ascribed to the nanoscale electrodemicrostructure formed directly on the electrolyte at a relativelylow temperature. These results reveal that the combination of differentimmiscible phases with different crystal structures and electrochemicalproperties could be a promising strategy to design highly efficientand durable air and fuel electrodes for SOFCs.La In this context, solid oxide cells (SOCs) areof great interest for electrical generation as they can convert thechemical energy of a wide variety of fuels, i.e., H2 andhydrocarbons, into electrical energy.3 Suchdevices can also operate reversibly in electrolysis mode to convertthe excess of electricity generated by renewable sources into hydrogen,offering numerous advantages compared to rechargeable batteries, suchas higher energy density, longer duration, and more flexibility.4The high demand forelectrical energy and the need for the protectionof the natural environment make the development of an effective infrastructureto produce energy from renewable sources, such as solar and wind,necessary. However, the electrical production from these sources isdiscontinuous and intermittent to meet the immediate demand;5 However, Ni-based anodes present mechanical stabilityissues upon redox cycling as well as carbon deposition and sulfurpoisoning when hydrocarbon fuels are employed.6 An alternative cell configuration, known as symmetrical solid oxidecells (SSOCs), where the same electrode material is used as both airand fuel electrodes, has gained great attention in the last few yearsbecause of its simpler fabrication process and improved chemical andthermomechanical stability to operate in fuel cell and electrolysismodes.7 Nevertheless, the major challengefor the development of SSOCs is to find a suitable electrode withhigh electrocatalytic activity and adequate long-term stability inboth oxidizing and reducing environments.9A conventional SOC is composed of a denseceramic ion conductorelectrolyte that separates two porous ceramic electrodes with differentcompositions, typically a mixed ionic–electronic conductorfor the air electrode and a Ni-cermet for the fuel electrode.3, SrFeO3, PrBa2O5+δ, and La0.4Sr1.6MnO4+δ.13 Among them, doped LaCrO3 is one of themost widely studied due to its high redox stability, resistance tosulfur poisoning, and carbon coking, as well as chemical compatibilitywith the traditional Zr0.84Y0.16O1.92 (YSZ) electrolyte. Different approaches have been developed to improvethe electrocatalytic properties of LaCrO3-based electrodes,including doping in the A- or B-site of the perovskite ,16 and the introduction of highly active catalytic elements,such as Ni and Ru, which are exsolved on the electrode surface uponexposure to reducing atmospheres.18Different symmetrical electrodes have been reported in the literature,including doped LaCrO19 However, nanostructuredelectrodes suffer from low thermal stability due to grain growth andcoarsening, limiting their application to the low temperature range(400–650 °C).20 Hence, themain challenge is to find nanostructured electrodes with sufficientperformance and durability for application in the intermediate temperaturerange (600–800 °C). This can be achieved using self-assemblednanocomposite electrodes consisting of a homogeneous and intimatemixture of two different materials. It has been found that a nanoscalecontact between multiple phases inhibits the grain growth and leadsto a large concentration of active sites for the electrochemical reactions.Moreover, the strong interphase interaction limits the thermal expansion,thus improving the mechanical properties.21 In this context, highly efficient air electrodes have been obtainedwhen mixed ionic–electronic conductors with good electrochemicalproperties are combined with an oxide-ion conductor, such as dopedceria and zirconia.22Anotheralternative strategy is the preparation of nanostructuredelectrodes by infiltration, spray-pyrolysis, and physical depositionmethods to increase the triple phase boundary (TPB), where the electrochemicalreactions take place.0.6Sr0.4MnO3−δ-Zr0.84Y0.16O1.92 (LSM-YSZ)23 and Sm0.5Sr0.5CoO3−δ–Ce0.8Sm0.2O1.9 (SSC-SDC).24 These nanocomposite cathodes showed an outstandingelectrochemical activity when compared to those obtained by physicallymixed powders. For instance, Shimada et al. reported a power densityover 3 W cm–2 at 750 °C for a screen-printedSSC-SDC nanocomposite.24 Several nanocompositecathodes have also been deposited directly by spray pyrolysis on theelectrolyte, simplifying the fabrication process and improving theadherence and integrity to the substrate when compared to ink-baseddepositions.26 This strategy has also been used to obtain Ni-YSZ anodes27 but, to the best of our knowledge, there areno studies on mixed oxide nanocomposites for fuel electrodes.In particular,spray pyrolysis has been used to prepare differentnanocomposite powder cathodes by a cosynthesis process, such as La0.98Cr0.75Mn0.25O3−δ–Ce0.9Gd0.1O1.95 (LCM-CGO)nanocomposites with different weight fractions of LCM by spray-pyrolysisdeposition. Mn-doped LaCrO3 was selected in this studydue to its high redox stability and improved performance for fueloxidation,28 while CGO is chemically compatiblewith LaCrO3-based electrodes and exhibits high ionic conductivity.Moreover, the perovskite is A-site-deficient and alkaline-earth-freeto prevent possible superficial phase segregation. The nanocompositeelectrodes were studied by a wide range of structural, microstructural,and electrochemical techniques to evaluate their potential use asboth air and fuel electrodes in solid oxide cells.Based on the idea that the combination of materials with differentconducting properties can be a promising approach to design new durableand efficient symmetrical electrodes, we have prepared, for the firsttime, La0.98Cr0.75Mn0.25O3−δ–Ce0.9Gd0.1O1.95, LCM-CGO)and different LCM contents  were prepared in a single step by spray-pyrolysis depositionfrom aqueous precursor solutions containing stoichiometric quantitiesof La(NO3)3·6H2O, Cr(NO3)3·9H2O, Mn(NO3)2·6H2O, Ce(NO3)3·6H2O, and Gd(NO3)3·6H2O. The metal concentration of thefinal solution was 0.02 mol L–1 in Milli-Q waterfor all compositions. Ethylenediaminetetraacetic acid (EDTA) witha concentration of 0.01 mol L–1 was added as a chelatingagent to prevent precipitate formation. For simplicity, the compositionof the different electrodes will be hereafter denoted as xLCM, where x represents the wt % of LCM. The precursorsolutions were sprayed with a flow rate of 20 mL min–1, atomized in a spray nozzle, and deposited on different substratetypes previously heated at 300 °C. The optimum deposition timeand nozzle–substrate distance were 1 h and 25 cm, respectively.After deposition, the layers were calcined in a furnace at 800 °Cfor 1 h in air with a heating/cooling rate of 2 °C min–1 to achieve crystallization.The electrodeswith nominal composition and La0.9Sr0.1Ga0.8Mg0.2O3−δ (LSGM) electrolytes. YSZ pelletsof 8 mm diameter were prepared from commercial powders supplied byTosoh, while LSGM powders were synthesized by the freeze-drying (FD)precursor method as described elsewhere.29 These powders were compacted into disks of 10 and 1 mm diameterand thickness, respectively, and sintered at 1400 °C for 4 hto reach a relative density greater than 98%.The nanocomposite layers werefirst deposited on amorphous quartz wafers of 2 × 4 cm30 LCM and Ce0.9Gd0.1O1.95  powders (1:1 wt %) were physically mixedby ball-milling with an organic polymeric vehicle (Decoflux). Theresulting ink was screen-printed onto the YSZ pellets and sinteredat 1100 °C for 1 h to achieve good adhesion to the electrolyte.For comparisonpurposes, LCM powders were also prepared from thefreeze-dried precursor method as described in detail elsewhere.α1,2 radiation). The patternswere analyzed with the X’Pert HighScore and GSAS programs forphase identification and structure analysis, respectively.32 The redox stability of the nanostructured electrodes was also investigatedafter successive annealing cycles in 5% H2–Ar andair atmospheres.The phase formation and crystal structurewere analyzed by X-ray powder diffraction (XRD) with an Empyrean PANalyticaldiffractometer  and high-angle annular dark-fieldscanning transmission electron microscopy (HAADF-STEM) in a TalosF200X (FEI-TEM). The grain size distribution was determined by thelinear intercept method using the Estereologia software.6 Hz with an AC perturbation of 50 mV in air, 5% H2–Ar, and wet 100% H2 (3 vol % H2O) atmospheres,as well as a function of the oxygen partial pressure to differentiatethe different rate-limiting steps involved in the oxygen reductionreactions. The effect of a direct current (DC) bias on the electrodeperformance was analyzed using a three-probe configuration with acircular working electrode of 0.20 cm2 and a Pt ring referenceelectrode surrounding the working electrode.34 The impedance spectra were collected with a Zahner XC potentiostat/galvanostat/impedanceanalyzer. The applied voltage was varied between 0 and 0.4 V in cathodicand anodic polarizations in both air and wet hydrogen atmospheresto evaluate the potential application of the electrodes in fuel celland electrolyzer modes. The spectra under dc bias were collected overtime until no change was observed. The impedance data were processedusing equivalent circuit models with the ZView software and distributionof relaxation times (DRT) with DRTtools.36Electrochemical impedance spectroscopywas used to determine theelectrode polarization resistance with a Solartron 1260 frequencyresponse analyzer in a two-electrode configuration at an open-circuitvoltage. The spectra were collected in the frequency range of 0.01–100.9Sr0.1Ga0.8Mg0.2O3−δ (LSGM) electrolyte at 300 °Cover an area of 0.20 cm2, followed by calcination at 800°C. A LSGM-supported cell was chosen instead of YSZ due to itshigher ionic conductivity, which is crucial to reduce the ohmic lossesof the cell. The electrolyte with a thickness and diameter of 300μm and 13 mm, respectively, was prepared from freeze-dried precursorsusing the methodology described in a previous report.30 The single cell was sealed on an alumina tube support witha glass-ceramic sealant . Current–voltageand impedance spectra were collected with a Zahner XC between 650and 800 °C using static air as oxidant and humidified H2 fuel (3% H2O), which was passed through a bubbler at20 °C. The H2 flow rate was set at 20 mL·min–1 and current–voltage (I–V) curves were obtained at a scan rate of 10 mV s–1.To evaluate the efficiency of the LCM-CGO composites in realoperatingconditions, the 50LCM electrode was deposited symmetrically by spraypyrolysis on a LaFm3̅m), while the diffraction peaks of 100LCM matched well with the theoreticalpattern of an orthorhombic perovskite-type structure (s.g. Pbnm). The patterns of the LCM-CGO nanocomposites show thediffraction peaks of two crystalline phases that are unequivocallyassigned to a fluorite and a perovskite-type compound. It is importantto remark that additional peaks attributed to secondary phases arenot detected.XRDpatterns of the nanocompositeelectrodes with different LCM contents and deposited on amorphousquartz substrates are shown in Rwp varies between 1.9 and 7.1%, thus confirming the accuracy of thefitting.The XRD patterns were analyzed by the Rietveld method to obtainfurther insights on the composition and crystal structure of the nanocompositeelectrodes. The usual Rietveld parameters were refined during theanalysis, i.e., scale factor, zero shift, background, peak-shape asymmetry,and preferential orientation. The cation site occupation factors werefixed and not refined. A representative Rietveld fitting for 50LCMis displayed in 3 for 100LCM at 800 °C, comparable to that obtainedfor the corresponding polycrystalline powders from freeze-dried precursors(235.121(3) Å3). However, the unit cell volume forLCM in the nanocomposite electrodes decreases slightly with the additionof CGO from 235.616(2) Å3 for 40LCM to 234.934(2)Å3 for 60LCM. A similar trend is observed for theCGO component: the unit cell volume increases from 159.202(4) Å3 for the blank CGO to 162.383(2) Å3 for 60LCM,suggesting that a minor cation interdiffusion occurs between CGO andLCM particles during the cosynthesis process. Since the ionic radiusof La3+ (1.16 Å) is larger than that of Ce4+ (0.97 Å), both in an eightfold coordination, the incorporationof La into the Ce site of CGO could explain the observed increaseof the cell volume.37 It has to be alsomentioned that minor cation interdiffusion between LCM and CGO isnot expected to have detrimental effects on the electrochemical properties.For instance, it is reported that Ce doping in LaCrO3−δ improves the performance due to the high catalytic activity of Ce4+/Ce3+ species for fuel oxidation.38 It is also worth noting that the phase quantification,determined by Rietveld analysis, is similar to the nominal one, furtherconfirming the composition of the nanocomposite electrodes Åectrodes 1.6 standard powder sample. This takes values of ∼10nm for CGO and 30 nm for LCM at 800 °C in the nanocomposite electrode. Nevertheless, the unitcell of LCM slightly expands after reduction in H2 dueto partial reduction of the B-site cations to lower oxidation stateswith the consequent increase of the ionic radii .The phase stability of nanocompositeelectrodes was investigatedat a high annealing temperature and under a reducing atmosphere. Noadditional diffraction peaks, attributed to secondary phases, wereobserved after treating the nanocomposite layers at 1000 °C for5 h, in accordance with previous findings for related materials 1.39 MorePbnm), CGO (s.g. Fm3̅m), and YSZ  or LSGM  substrates.Representative Rietveld plots for 50LCM are displayed in Figure S1c,d and the structural parameters aresummarized in Similar results are observed for the nanocompositeelectrodes depositedon polycrystalline YSZ and LSGM electrolytes. In this case, threecrystalline phases are considered to fit the XRD patterns, i.e., LCM.Cross-sectional SEM images of the cell show a porousmorphologyfor both 100LCM and 50LCM 3a,b. The2 atmosphere at the open-circuit voltage areshown in Impedance spectraof the different nanocomposite electrodes in symmetrical cell configurationin air and H42 Two contributionsdenoted as HF and LF at high and low frequencies, respectively, werediscernible in both oxidizing and reducing atmospheres for all electrodecompositions . According tothe DRT results, the spectra were adequately fitted using an equivalentcircuit consisting of two RQ elements in series to simulate the highand low frequencies, HF and LF contributions, respectively, to theelectrode polarization. In addition, a series resistance Rs and an inductance L are also includedto take into account the ohmic contribution of the electrolyte andthe inductive effects of the electrical connections, respectively.44 In the case of the nanocomposite electrodes, the TPB length is significantlyincreased and higher capacitances are obtained, about 2 and 20 mFcm–2 for the HF and LF processes, respectively,regardless of the electrode composition of the nanocomposite. Regardingthe resistance of the HF and LF responses, it is important to highlightthat both contributions decrease significantly for the nanocompositeelectrodes although 50LCM composition exhibits the lowest values . In addition, the activation energiesare similar with values of ∼1.4 and 1.7 eV for HF and LF processes,respectively.In oxidizingconditions, the screen-printed 50LCM_SP has capacitancevalues of about 0.01 and 0.1 mF cm2–Ar atmosphere, theLF response isthe dominant contribution to the total polarization resistance. Thisprocess has a large capacitance of 0.2 F cm–2 andits resistance decreases abruptly in pure H2 from 1.67to 0.11 Ω cm2 with a hydrogen partial partial dependenceof (pH2)0.92, suggesting that it is possiblyattributed to gas diffusion and concentration limitations .45 In contrast,the HF response with a capacitance of 15 mF cm–2 is less dependent on the H2 concentration and is associatedwith charge transfer processes at the electrode/electrolyte interface.In a 5% HFigure S6). The impedancespectra are comprised of two processes (HF and LF) in both air andhydrogen atmospheres with capacitances and frequency values similarto those observed for the YSZ electrolyte. Thus, the same electrochemicalprocesses seem to be involved in both LSGM and YSZ electrolytes. However,the total polarization resistance decreases slightly due to the higherionic conductivity of LSGM compared to YSZ.Electrode polarization studies were also performed on the LSGMelectrolyte and the results were similar to those observed for theYSZ electrolyte .In addition, the activation energy of the polarization resistanceis lower for the nanocomposite electrodes with values of 1.9 and 1.5eV for 100LCM and 50LCM, respectively, implying that the oxygen reductionreaction kinetics is improved upon CGO addition.The total polarizationresistance, ectively 4c. It is2 is used instead of diluted hydrogen(5% H2–Ar), which is attributed to a drastic reductionof the LF contribution due to surface electrode processes. The optimalcomposition with the lowest polarization resistance is found for 50LCM,i.e., 0.09 Ω cm2 at 750 °C compared to 0.29and 1.8 Ω cm2 for 60LCM and 100LCM, respectively.It is also worth noting that these values are lower than those reportedfor the most efficient symmetrical electrodes (0.75Sr0.25Cr0.5Mn0.5O3−δ (0.30Ω cm2 in H2 at 900 °C)10 and Sr2Fe1.5Mo0.5O3−δ (0.45 Ω cm2 in H2 at 800 °C),46 and relatedanode materials with exsolved metal particles, i.e., La0.75Sr0.25Cr0.5Mn0.3Ni0.2O3−δ (1.1 Ω cm2 at 800 °Cin 5% H2)47 and Sm0.8Sr0.2Fe0.8Ti0.15Ru0.05O3−δ (0.2 Ω cm2 at 800 °Cin 5% H2).48 In addition, theactivation energy values vary between 1.0 eV for 50LCM and 1.2 eVfor 100LCM, and as expected, they are lower than those obtained inair atmosphere.In a reducingatmosphere, important differences are observed dependingon the hydrogen concentration and CGO content in the nanocompositeelectrodes 4d. Firstectrodes 2, such a2) to identify the different processesinvolved in the oxygen reduction reaction m−,where m provides information about the type of speciesinvolved in each process.58Since 50LCMexhibits improved performance as both fuel and airelectrodes, this composition was further characterized by other electrochemicaltechniques to obtain further insights on the nature of the electrochemicalprocesses. The impedance spectra were acquired at different oxygenpartial pressures . However, when an anodic polarizationis applied, the polarization resistance increases up to 0.55 Ωcm2 at +0.4 V. The HF process, assigned to the charge transferprocess, remains constant under dc bias in both anodic and cathodicpolarizations. In contrast, the LF process decreases under cathodicpolarization and increases under anodic polarization. A similar behaviorhas been observed for La0.3Sr0.7Fe0.7Cr0.3O3−δ and Sr1.8Ba0.2Fe1.5Mo0.5O6−δ under anodic polarization; however, additional studies at differentpH2 and pH2O values are needed to better understandand identify the different processes involved in the hydrogen oxidationreaction (HOR) and hydrogen evolution reaction (HER) in these nanocompositeelectrodes.67The measurements performed in wet 100% H2 as fuel, are displayed in Rs increases with decreasing temperature from0.32 to 0.50 Ω cm2 at 800 and 700 °C, respectively,which are in good agreement with the estimated values for a 300-μm-thickLSGM electrolyte .Finally,a short stability test was carried out at 750 °Cfor 100 h and the peak power density of the cell, obtained by applyinga constant voltage of 0.55 V over time, remained almost constant 7c. Moreo4+ to Ce3+ reduction.69 (vi) Moreover, these nanocomposite electrodes are alkaline-earth-free,making them less susceptible to carbonation and surface phase segregation,which usually deteriorate the electrochemical properties after long-termoperation.70 (vii) Moreover, recently,nanoscale La0.9Sr0.1CrO3−δ has been reported to exhibit an enhancement of the oxygen ion diffusivityand surface exchange coefficients compared to the bulk.71 Thus, the combination of all of these propertiesprovides suitable nanocomposites to operate simultaneously as bothair and fuel electrodes and could be the key for the development ofnew symmetrical electrodes for SOCs.In summary, better electrochemical properties ofLCM-CGO nanocompositesare associated with several factors: (i) the low preparation temperature,which minimizes the chemical reactivity at the electrolyte/electrodeinterface. (ii) The self-assembled nanocomposite electrodes exhibita strong interphase interaction, reducing the thermal expansion andimproving the physical compatibility and adherence to the electrolyte.(iii) The low particle size of the nanocomposite electrodes significantlyincreases the TPB length, and consequently, the efficiency. (iv) Thesynergetic effect between LCM and CGO with high electronic and ionicconductivities, respectively. (v) Ceria is also known to release morelattice oxygen when decreasing the particle size, and it exhibitshigh mixed ionic–electronic conductivity under reducing conditionsdue to Cex·La0.98Cr0.75Mn0.25O3−δ–Ce0.9Gd0.1O1.95 (x = 0–60 wt %) were preparedfor the first time in a single cosynthesis step by spray-pyrolysisdeposition, reducing the fabrication time and costs compared to atraditional and physically mixed composite electrode deposited bythe screen-printing method. The structural and microstructural evolutionwith the annealing temperature confirmed that CGO and LCM phases arepractically immiscible in the temperature range studied up to 1000°C. Moreover, the nanocomposite electrodes presented high redoxstability, which makes them suitable for both air and fuel electrodes.Nanocompositeelectrodes with a nominal composition of 2 in air at 750 °C.The strong interphase interaction between the CGO and LCM nanoparticleslimited the grain growth rate and improved the thermal compatibilitywith the electrolyte. Interestingly, the nanometric particle sizeis retained at high annealing temperatures with values as low as 10and 50 nm at 800 and 1000 °C, respectively. The higher nanoscalecontact between the CGO and LCM particles led to larger concentrationof catalytic active centers compared to a screen-printing electrode,and consequently, the polarization resistance was greatly reducedfrom 2.05 to 0.29 Ω cm–2 at 800 °C, higher than that reported previously for a LaCrO3-based electrode. Thus, the preparation of nanocompositesby a cosynthesis method is a promising strategy to combine the electrochemicalproperties of multiple phases to design new highly efficient and durablesymmetrical electrodes for application in fuel cell and solid electrolyzercells.The 50LCM symmetrical electrode over a LSGM-supported cell showeda remarkable maximum power density of 570 mW cm


Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the United States and the second most common indicator for liver transplantation . NAFLD iFree cholesterol (FC) has been shown to disrupt mitochondrial function and induce hepatocyte death, contributing to NAFLD progression . In SS, Recently, it has been proposed that NAFLD is a mitochondrial disease because besides being the cell's power plants, mitochondria are also responsible for cholesterol transport, fatty acid oxidation, and generation of reactive oxygen species. Mitochondrial dysfunction renders liver cells susceptible to damage and extracellular risk factors by disrupting intracellular metabolism and signal transduction .ATPase Family AAA Domain Containing 3A (ATAD3A) is a ubiquitous mitochondrial transmembrane ATPase , 8 and pTo gain insight into the function of ATAD3A in the progression of NAFLD, we characterized ATAD3A expression in liver samples from rats with NASH along with matching controls and liver specimens from patients with SS, NASH, cirrhosis, or normal tissue. ATAD3A was found to be upregulated with the progression of NAFLD, revealing the possible involvement of ATAD3A. Moreover, loss of ATAD3A increased FC and TG accumulation under FC overload in Huh7 cells and blocked autophagosome degradation in both Huh7 cells and fresh human hepatocytes isolated from humanized liver chimeric mice (HLCM). Mitophagy was largely inhibited after the loss of ATAD3A under FC overload in Huh7 cells. ATAD3A KO Huh7 cells under FC overload also showed reduced mitochondrial respiration. Collectively, our results indicated that ATAD3A plays an essential role in the progression of NAFLD.A). To see if this is applied to humans, we stained liver sections from normal individuals and patients with SS, NASH, or cirrhosis with anti-ATAD3A antibody. The human liver samples were first blind-scored using the NAFLD activity score system by a pathologist, regardless of gender. Then, 12 samples were randomly picked as representative of different liver stages by NAS grades of normal, steatosis, NASH, and cirrhosis (n = 3 for each stage) for further analysis. The results showed that in patients with SS, NASH, and cirrhosis, the expression of ATAD3A in the liver was increased compared to normal . Collectively, this suggests that ATAD3A may either play a role in the origin of NAFLD or be part of the disease phenotype.To assess the expression levels of ATAD3A in the progression of NAFLD, we fed Sprague Dawley rats a methionine-choline deficient (MCD) diet for 8 weeks to induce a NASH phenotype . Chow-feFig. 1A).To examine the role of ATAD3A in the progression of NAFLD, CRISPR-Cas9 was performed to generate an ATAD3A KO Huh7 cell line. The loss of ATAD3A was demonstrated by immunoblot analysis A.Figure B).Confocal microscopy imaging demonstrated that most of ATAD3A colocalized with the mitochondrial marker MitoTracker in WT Huh7 cells. Image analysis showed a Pearson’s coefficient of 0.937 and a Mander’s coefficient of 0.87 (fraction of ATAD3A overlapping with MitoTracker). ATAD3A fluorescence was not seen in the ATAD3A KO Huh7 cells, confirming the loss of ATAD3A B.C). Notably, transmission electron microscopy (TEM) images revealed a rounded mitochondrial structure and disrupted mitochondrial cristae in the ATAD3A KO Huh7 cells (D), which is consistent with the mitochondrial cristae alteration in MA-10 cells in response to ATAD3A siRNA . Interestingly, FC levels in KO cells were significantly higher than WT under FC overload . Treated Huh7 cells were also stained with Filipin, a dye commonly used to stain FC, and observed under fluorescent microscopy. As shown in Figure 3B, the blue Filipin puncta were more abundant and larger in KO cells than WT under FC overload, as well as control conditions. The Filipin positive signals were then quantified and normalized to the number of nuclei. As shown in Figure 3C, the % area stained with Filipin per cell was significantly higher in KO cells than WT under FC overload and control conditions.Since FC rather than total cholesterol contributes to the progression of NAFLD , we induFig. 3D). HMGR protein levels were suppressed after loss of ATAD3A under control conditions, but the difference between WT and ATAD3A KO cells was less obvious under FC overload (F). ATAD3A KO tended to suppress mRNA expression of liver X receptor alpha (LXRA), a gene involved in cholesterol metabolism, but protein levels remained stable under FC overload (E and F). Taken together, ATAD3A KO induces FC escalation upon FC accumulation without causing significant change to cholesterol synthesis and metabolism under FC overload, suggesting that ATAD3A may affect other pathways involved in cholesterol regulation, such as cholesterol secretion.To examine the mechanism underlying ATAD3A KO deteriorated FC accumulation under FC overload, we next tested whether cholesterol synthesis or metabolism was altered after the loss of ATAD3A. The quantitative PCR (qPCR) results did not show obvious differences in mRNA levels of HMG-CoA reductase (HMGR), the rate-limiting enzyme for cholesterol synthesis, between KO and WT under FC overload, even though ATAD3A KO significantly upregulated mRNA expression of HMGR in controls D. HMGR poverload F. ATAD3Aoverload , E and Fde novo biosynthesis (A and B). At the protein level, FAS was moderately downregulated in ATAD3A KO cells under FC overload, whereas no obvious change was seen for SREBP1c (C and D). These data suggest the lower free fatty acids production in the ATAD3A KO cells under FC overload. Interestingly, however, ATAD3A KO significantly increased TG accumulation under FC overload and control conditions in Huh7 cells, suggesting that lipolysis may be inhibited in ATAD3A KO cells (E). Since TG is stored in lipid droplets, we stained cells with Nile red, a dye used to stain intracellular neutral lipids in lipid droplets. Image analysis showed an increased number of small lipid droplets in ATAD3A KO cells compared to WT Huh7 cells under control and FC overload conditions (A and B).Next, we are interested to see if ATAD3A KO alters TG homeostasis. To this end, qPCR was performed to measure the expression of lipogenic genes in Huh7 cells, including sterol regulatory element-binding transcription factor 1 (SREBP1c) and fatty acid synthase (FAS). SREBP1c is the key transcription factor that masters the activation of genes for fatty acid synthesis, whereas FAS is the rate-limiting enzyme for fatty acid ynthesis . It was ynthesis , A and B SREBP1c , C and DKO cells E. Since nditions , A and BF). Moreover, terminally differentiated human hepatocytes were freshly isolated from the liver of HLCM and subjected to either ATAD3A siRNA to knock down ATAD3A or universal siRNA as the control followed by the same treatment for Huh7 cells. Consistently, FC overload suppressed PLIN2 protein expression, whereas ATAD3A knockdown (KD) by siRNA increased PLIN2 expression back to a normal level in human hepatocytes . Taken together, ATAD3A KO induces TG accumulation under FC overload as well as control conditions via inhibition of lipolysis.Being a negative regulator for lipolysis, Perilipin 2 (PLIN2) is a protein associated with lipid droplets' (LDs) surface, stabilizing LDs and protecting neutral lipids from enzymatic metabolism. Liver-specific ablation of PLIN2 reduced neutral lipids and liver steatosis in mice fed with MCD diet, whereas PLIN2 overexpression in HepG2 cells triggered neutral lipid accumulation , 22. As Fig. 4H–J).Despite less fatty acid synthesis, as suggested by the downregulation of lipogenic gene expression following the loss of ATAD3A, ATAD3A KO significantly upregulated mRNA and protein expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme for fatty acid oxidation, and displayed the tendency to upregulate mRNA but not protein of peroxisome proliferator activated receptor alpha (PPARA), one of the upstream regulators of CPT1A, under FC overload in Huh7 cells, which may be the result of compensation for less free fatty acids store and lack of energy production , H–J.A, FC overload dramatically upregulated the expression of microtubule-associated protein 1A/1B-light chain 3-II (LC3-II), a wildly-used marker for autophagy, and ATAD3A KO pushed this process further. The mRNA expression levels of autophagic adapter, p62, was also significantly increased in ATAD3A KO Huh7 cells compared to WT under FC overload and control conditions despite only a slight increase in p62 protein levels following the loss of ATAD3A (A and B). To further confirm these results, LC3-II and p62 protein levels were also measured in human hepatocytes with ATAD3A KD by siRNA under the same treatment conditions as Huh7 cells. Consistently, ATAD3A KD dramatically upregulated LC3-II and p62 protein levels in human hepatocytes (C). Furthermore, more abundant and larger autophagolysosomes were observed in ATAD3A KO Huh7 cells than in WT under FC overload, as shown in the TEM images (D).Because autophagy plays an essential role in the progression of SS and is inducible by FC overload , 24, we Figure 5f ATAD3A , A and Batocytes C. FurtheM images D.Figure E, 3-MA effectively reduced LC3-II levels increased by FC overload in WT cells , suggesting that FC overload induces autophagosome formation. Furthermore, the presence of bafilomycin A1 further increased LC3-II induced by FC overload in WT cells , indicating that autophagic degradation is unaffected under FC overload (F). Therefore, FC overload increases autophagic flux.As known, the upregulation of LC3-II and accumulation of autophagolysosomes can be attributed to either elevation in autophagosome formation or blockade of autophagosome degradation. To further tease out which process was affected by the loss of ATAD3A, we pretreated cells with autophagy inhibitors 3-methyladenine (3-MA), a PI3K inhibitor, or bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, for 1 h before cholesterol and 58035 exposure. As shown in Figure 5overload F. TherefE, lane 1 versus 5 and 3 versus 7), the upregulation of LC3-II and p62 in ATAD3A KO cells was barely reversed by 3-MA, suggesting that ATAD3A KO does not induce autophagosome formation . On the other hand, the upregulation of LC3-II in KO cells over WT was lost in the presence of bafilomycin A1 under FC overload or control conditions , suggesting that autophagolysosomal activity was reduced and autophagosome degradation was blocked after loss of ATAD3A. In contrast, the transient overexpression of ATAD3A after FC accumulation with cholesterol and 58035 treatments enhanced the autophagic degradation evidenced by increased LC3-II and degraded p62 compared to WT . Taken together, these data indicate that ATAD3A KO blocks autophagosome degradation under FC overload and control conditions, reducing cell survival.It has been reported that elevated autophagy is a protective mechanism for cells under FC overload and inhibition of autophagy enhanced cell apoptosis . TherefoA, the expression of PTEN-induced kinase 1 (PINK1), the initial adapter for PINK1-Parkin mediated mitophagy, was dramatically downregulated after the loss of ATAD3A under FC overload and basal conditions, suggesting that ATAD3A is essential for the PINK1-Parkin–mediated mitophagy and loss of ATAD3A will inhibit mitophagy. Consistently, MitoTracker green, an indicator of mitochondrial mass, was significantly increased after the loss of ATAD3A under both control conditions and FC overload, suggesting accumulation of mitochondria after deletion of ATAD3A (B). The upregulation of mitochondrial proteins cytochrome c oxidase subunit 4 (COX IV) and translocase of outer mitochondrial membrane 20 (TOM20) further confirmed the increased mitochondrial mass in ATAD3A KO cells under FC overload (A).Since ATAD3A is located in inner mitochondrial membrane, we examined whether mitophagy, a selective autophagy process of mitochondria, was affected after the loss of ATAD3A under FC overload. As shown in Figure 6f ATAD3A B. The upoverload A.Figure C–F).The analysis of mitochondrial function using a Seahorse analyzer was undertaken to see whether the accumulation of mitochondria after ATAD3A deletion is beneficial to the energy supply of cells. The results showed that basal respiration and ATP production significantly dropped after the loss of ATAD3A under FC overload, even though only a decline trend was seen for maximum respiration, suggesting that ATAD3A KO tends to accumulate dysfunctional mitochondria in cells under FC overload , C–F.G, although ATAD3A KO did not affect complex II and complex III expression, complex IV and complex I were obviously upregulated in ATAD3A KO cells compared to WT under both control conditions and FC overload. Intriguingly, however, complex IV, the ATP synthase responsible for ATP generation, was drastically downregulated in ATAD3A KO cells compared to WT under control conditions and FC overload. These data indicate that ATAD3A is vital for the normal balance and function of oxidative phosphorylation complex enzymes, and loss of ATAD3A reduces cellular ATP production partially through disruption of the oxidative phosphorylation complex. The highly damaged mitochondrial structure and disrupted mitochondrial content in the ATAD3A KO cells under FC overload can also be seen by TEM (H).To examine whether the compromised mitochondrial respiration and ATP production after loss of ATAD3A were linked to the mitochondrial oxidative phosphorylation complex, the expression of five oxidative phosphorylation complex enzymes was measured. As shown in Figure 6n by TEM H.Even though ATAD3A has been reported to be involved in cholesterol transport, the function of ATAD3A in NAFLD remains unclear. Herein, we show the upregulation of ATAD3A in the progression of NAFLD in livers from humans and rats. Since it has been shown that cholesterol homeostasis disruption and FC overload contribute to NAFLD pathology, it is more relevant to investigate the function of ATAD3A under FC overload .Loss of ATAD3A upregulated LC3-II and p62 under FC overload in Huh7 cells and HLCM-derived human hepatocytes and downregulated PINK1 expression, suggesting ATAD3A KO under FC overload blocks autophagosome degradation and mitophagy. Furthermore, loss of ATAD3A impaired mitochondrial basal respiration and ATP production under FC overload in Huh7 cells.Given that p-mTOR is upregulated in NASH-related cirrhosis  and mTORIn Huh7 cells, we found that after the loss of ATAD3A, mitochondria became dilated, and cristae were disrupted, which is consistent with previous findings . The altUnexpectedly, even though the loss of ATAD3A induced significantly higher FC accumulation under FC overload, neither key enzymes in cholesterol synthesis nor genes involved in cholesterol metabolism showed significant changes. Loss of ATAD3A significantly upregulated HMGR mRNA expression under control conditions but did not impact its expression under FC overload, even though there was a decline in HMGR mRNA expression in ATAD3A KO cells under FC overload. HMGR protein levels were downregulated rather than upregulated to contribute to the FC increase after loss of ATAD3A under FC overload. Furthermore, LXRA expression was not significantly affected by the loss of ATAD3A under FC overload either, even though a declining trend was seen, suggesting that ATAD3A may impact cholesterol export from cells to affect the intracellular cholesterol pool.It is known that ATAD3A mutation or silencing alters autophagic levels , 28. In The upregulation of PLIN2 after the loss of ATAD3A indicates that suppressed lipolysis contributes to the TG accumulation. PLIN2 protects neutral lipids from lipolysis when coated at the surface of LDs and is identified as a therapeutic target for NAFLD. It has been shown that loss of PLIN2 in hepatocytes partially protected Western diet-fed mice from steatosis and largely from NASH, whereas overexpression of PLIN2 elevates both TG and LDs , 31. Thevia degradation of LDs. In contrast, it has also been proposed that elevated autophagy may also contribute to the TG storage by degradation of cellular materials to provide FA for TG synthesis in hepatocytes . The studies in this work abide by the Declaration of Helsinki principles. A detailed description of the human liver samples used was recently published and is summarized in For MCD diet studies, male rats aged 12 weeks old were fed either regular rat chow or a MCD diet  for 8 weeks. The rats were divided into 2 groups (chow and MCD n = 3). The body weight was recorded every week. After 2 months, the rats were harvested, and fresh liver tissue samples were placed into micro-Eppendorf tubes and stored in −80 °C for protein measurement. The rats were bred and maintained in accordance with protocols approved by the IACUC of the University of Southern California.2 incubator. The medium was replaced every 3 days if not otherwise stated.Huh7 cells were purchased from JCRB Cell Bank  and maintained in Dulbecco's Modified Eagle Medium (DMEM)  supplemented with 10% of fetal bovine serum (FBS)  and 1% of Penicillin-Streptomycin  at 37 °C and 5% CO2 incubator after isolation. The medium was replaced every 3 days within 1 week after isolation. Then the medium was replaced to DMEM  supplemented with 10% of FBS  and 1% of Penicillin-Streptomycin  for continued culture until experimental treatments.Human hepatocytes were cultured in DMEM  supplemented with 20 mM Hepes , 10% of FBS , 1% of Penicillin-Streptomycin , 15 μg/ml L-Proline , 0.25 μg/ml insulin , 50 nM dexamethasone , 5 ng/ml epidermal growth factor , 0.1 mM L-ascorbic acid 2-phosphate (Asc-2P) , 2% DMSO  at 37 °C and 5% COATAD3A CRISPR KO Vector ALL In One (set of 3 targets)  encoding sgRNA targeting ATAD3A exon 7 sequence 5′-CGAGCAGATCCGCCTGAAGG-3′ and Cas9 was transfected into Huh7 cells followed by 2 μg/ml puromycin  selection. Transfected Huh7 cells were then redistributed onto a 100-mm culture dish to allow the formation of single cell colonies. After culturing for one month, single cell colonies were picked up under the microscope and transferred to 6-well plates. When single cell colonies reached 80% confluency, protein was extracted and subjected to immunoblotting to detect the loss of ATAD3A.6/ml. One hour before transfection, the medium was replaced with fresh complete medium. A liquid mixture of lipofectamine 3000 reagent and lentiviral vector was added to cells and incubated for 24 h. Then the medium was replaced with DMEM supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight, followed by the treatment with cholesterol and 58035 for 24 h. The cell pellet was then collected and subjected to immunoblotting.Customized lentiviral vector expressing ATAD3A isoform 2 and control vector were purchased from VectorBuilder. Lipofectamine 3000 Transfection Reagent  was used for transfections following manufacturer’s instructions. Briefly, Huh7 cells were seeded onto 6-well plates at a density of 0.4 × 106/ml, 0.2 × 106/ml, or 0.1 × 106/ml in 6-well plates, 12-well, and 24-well plates or 96-well plates, respectively, with DMEM supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  and incubated overnight. Cholesterol–methyl-β-cyclodextrin  and Sandoz 58035  were added to the medium to a final concentration of 100 μM and 10 μg/ml, respectively, and incubated for 24 h. 0.01% DMSO  was added to cells as the control. The pretreatment time was 1 h for both 3-MA  (2 mM) and bafilomycin A1  (100 nM).Huh7 cells or human hepatocytes were seeded at a density of 0.4 × 10In Situ Mounting Medium with DAPI . Images were taken with a ZEISS LSM 880 Confocal Microscope. The colocalization analysis was performed using ImageJ v1.52a.Huh7 cells were seeded on coverslips in DMEM  supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight prior to treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Cells were then incubated with phenol red-free DMEM  containing 100 nM MitoTracker Deep Red FM  for 30 min at 37 °C. After washed with PBS, cells were immobilized with 4% paraformaldehyde  for 10 min, incubated with 0.1% of Triton-X  for 10 min, and blocked with PBS  containing 5% of donkey serum  and 0.5% of bovine serum albumin (BSA)  for 30 min. Cells were then incubated with primary antibody against ATAD3A  with a dilution of 1:400 at 4 °C overnight, followed by the addition of the Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary antibody, Alexa Fluor 568  for 1 h. Cells were stained with Duolink Human hepatocytes were isolated from HLCM as described previously. In brief, HLCM were produced by an injection of commercially available cryopreserved primary human hepatocytes  into the spleen of severe combined immunodeficiency mice with albumin enhancer/promoter driven cDNA-urokinase type plasminogen activator transgene expression as previously described . The useRNAi of ATAD3A was conducted using Lipofectamine RNAiMAX Transfection Reagent  following the manufacturer's instructions. Briefly, human hepatocytes were grown in culture medium overnight before the day of transfection. One hour before transfection, the medium was replaced with fresh complete medium. The mixture of Lipofectamine RNAiMAX Reagent and human ATAD3A siRNA  was then added and incubated for 24 h. The MISSION siRNA Universal Negative Control #1  was used as control siRNA.g at 4 °C for 30 min to remove cell debris. Supernatants were taken out, and protein concentrations were determined using Pierce BCA Protein Assay Kit . Loading buffer  was added to cell extracts, and the mix was heated to 95 °C for 10 min in a digital heatblock (VWR). Samples were loaded onto a protein gel  and run under 100 V for 1 h. The protein in the gel was transferred to polyvinylidene difluoride membrane  under 80 V for 2 h. The membrane was blocked with 5% of BSA for 30 min and incubated with primary antibodies against HMGR , LXRA , SREBP1c , FAS , PLIN2 , PPARA , CPT1A , ATAD3A , LC3-II , p62 , PINK1 , Parkin , COX IV , TOM20 , Total OXPHOS , GAPDH , or alpha-Tubulin  at 4 °C overnight. After washing with PBST ), 5% of BSA with Goat anti-Rabbit HRP Secondary antibody  or Goat anti-Mouse HRP Secondary antibody  was added to the membrane and incubated at RT for 1 h. The images were taken by Azure c600 biosystem (Azure Biosystems). Following immunoblot analysis with a specific antibody, membranes were stripped of the antibodies with Stripping Buffer  and reblotted with an antibody targeting an internal control, for example, GAPDH or alpha-Tubulin. In search of more than one antigen of different molecular weights, separated and blotted on the same membrane, the membrane was cut, and each strip blotted with antibodies specific to antigens and internal control, for example, GAPDH or alpha-Tubulin.Cells were harvested at 5000 rpm for 5 min and washed with PBS. Lysis buffer  was added to the cell pellet, and cell extracts were incubated on ice for 30 min. Cell extracts were then centrifuged at 17,2006 cells were harvested, pelleted, and fixed in 1/2 strength Karnovsky solution, containing 2.5% glutaraldehyde and 2% paraformaldehyde in PBS, overnight at 4 °C. The cell pellet was rinsed in a 0.1 M cacodylate buffer and postfixed in 2% aqueous OsO4/0.2 M cacodylate buffer for 2 h at 4 °C, followed by several rinses with 0.1 M cacodylate buffer. An EM block was stained with saturated uranyl acetate overnight at 4 °C. After rinsing with 0.1 M sodium acetate buffer, the cells were dehydrated through a gradient series of 30%–100% ethanol, placed in 100% propylene oxide, and infiltrated in a 1:1 mixture of propylene oxide:polybed 812 epoxy resin  overnight at room temperature. After several changes of 100% resin over 24 h, the pellet was embedded in Quetol 812 . Areas containing cells were block mounted and cut into 70-nm sections with a Leica EM UC6 Ultramicrotome . The sections were stained with uranyl acetate (saturated aqueous solution) and lead citrate and examined with a transmission electron microscope .1 x 106 cells were pelleted at 5000 rpm for 5 min and washed with PBS. Two hundred microliters of chloroform:isopropanol:IGEPAL CA-630 (7:11:0.1) were added, and samples were homogenized at an oscillation frequency of 60/s for 1 min in TissueLyser II (Qiagen) to extract the cholesterol. Cell lysates were centrifuged at 17,200g for 30 min to remove insoluble material. The supernatant was taken out and air dried at 50 °C in a speed vacuum for 1 h. The dried lipids were redissolved in 250 μl of assay buffer. For the assay, 50 μl of each sample along with 50 μl reaction mix were set up in duplicate using 96 well plates and incubated at 37 °C for 1 h. The absorbance at wavelength of 570 was detected by a multimode plate reader (PerkinElmer VICTOR X5). The amount of cholesterol was normalized to the protein concentration determined by Pierce BCA Protein Assay Kit .The cholesterol was quantified using Cholesterol Quantitation Kit  following the manufacturer's instructions. Briefly, 1 × 10Huh7 cells were seeded on coverslips in DMEM  supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight prior to treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Huh7 cells were washed with PBS and immobilized by adding 4% (wt/vol) paraformaldehyde for 10 min. Cells were then washed three times with PBS and incubated with 0.1% of Triton-X for 10 min. The cells were then washed three times again in PBS and blocked with PBS containing 5% of donkey serum and 0.5% BSA for 30 min. After washing three times with PBS , cells were incubated with 0.05 mg/ml Filipin  and primary antibody against LaminB1  at a dilution of 1:400 at a room temperature for 2 h. Cells were again washed three times with PBS and incubated with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary antibody, Alexa Fluor 488  for 1 h. After another three washes with PBS, the cells were mounted with Fluoromount-G Slide Mounting Medium . The images were visualized and captured with fluorescent microscopy .RNA was isolated using Quick-RNA Miniprep Plus Kit . RNA was reverse transcribed to cDNA using PrimeScript RT Master Mix  , following the manufacturer’s instructions, by Mastercycler nexus X2 (Eppendorf). The qPCR was conducted by Biotek. The internal control is human ribosomal protein S18 (Rps18). The expression was normalized to S18 mRNA. The primer sequences were as follows: HMGR-F: 5′-AGTGAGATCTGGATCCAA-3'; HMGR-R: 5′-GATGGGAGGCCACAAAGAGG-3'; LXRA-F: 5′-TCTGGACAGGAAACTGCACC-3'; LXRA-R: 5′-CCGCAGAGTCAGGAGGAATG-3'; SREBP1C-F: 5′-TGCTGCAGGAGTCTGAGAGA-3'; SREBP1C-R: 5′-CTGGACCAGACTCTGCCTTG-3'; FAS-F: 5′-GCAAGCTGAAGGACCTGTCT-3'; FAS-R: 5′-AATCTGGGTTGATGCCTCCG-3'; PLIN2-F: 5′-TGATGGCAGGCGACATCTAC-3'; PLIN2-R: 5′-AAAGGGACCTACCAGCCAGT-3'; PPARA-F: 5′-TGGGAAGGCAGCGTTGATTA-3'; PPARA-R: 5′-CTGGCAGTTCCAGTCCAGAT-3'; CPT1A-F: 5′-GTTCTCTTGCCCTGAGACGG-3'; CPT1A-R: 5′-TTTCCAGCCCAGCACATGAA-3'; p62-F: 5′-AGCGTCAGGAAGGTGCCATT-3'; p62-R: 5′-TTCTCAAGCCCCATGTTGCAC-3'; S18-F: 5′-ATTAAGGGTGTGGGCCGAAG-3’; and S18-R: 5′-TGGCTAGGACCTGGCTGTAT-3’.6 cells were harvested and homogenized in 500 μl solution of 5% Nonidet P 40 Substitute . Samples were then heated to 100 °C in a digital heatblock (VWR) for 2 to 5 min until the Nonidet P 40 becomes cloudy, followed by cooling temperature to room temperature. Samples were centrifuged at 17, 200g for 5 min to remove insoluble material at room temperature. The supernatant was collected followed by the addition of lipase and master reaction mix in the kit. The absorbance at 570 nm was measured by Synergy H1 multiplate reader (BioTek). The amount of TG was normalized by the protein concentration determined by Pierce BCA Protein Assay Kit .TGs were quantified using Triglyceride Quantification Kit  following the manufacturer's instructions with minor modifications. Briefly, 1 × 106 cells/ml) were seeded on coverslips in DMEM  supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight. Cells were treated with 100 μM cholesterol and 10 μg/ml 58035 for 24 h and fixed with 4% (wt/vol) paraformaldehyde for 10 min and washed with PBS for three times. Glycine  was then added to quench the cells for 10 min at room temperature. After washing with PBS for three times, the cells were incubated with 10 μg/ml Nile red  at room temperature for 10 min. After completion of staining, the Nile red was removed, and cells were washed with PBS. Cells were then stained with Duolink In Situ Mounting Medium with DAPI . Images were visualized and captured by fluorescent microscopy .Huh7 cells  supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight before treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Cells were then washed with PBS and incubated with phenol red-free DMEM  containing 100 nM MitoTracker Green FM  for 30 min to allow for the internalization of MitoTracker into cells. Cells were washed with PBS three times and mounted with Fluoromount-G Slide Mounting Medium . The images were visualized and captured with fluorescent microscopy .The MTT assay was conducted using cell proliferation kit I (MTT) , following the manufacturer's instructions. Briefly, cells were seeded at a density of 10,000 cells/well in a 96-well plate followed by 24-h incubation with treatments. After incubation, 10 μl of MTT labeling reagent was added to each well and incubated for 4 h in 37 °C. Hundred microliters of solubilizing solution was subsequently added and incubated overnight in 37 °C incubator. The absorption at 595 nm was measured by a multimode plate reader (PerkinElmer VICTOR X5).Human hepatocytes from HLCM were isolated and seeded on coverslips followed by the RNAi for ATAD3A using Lipofectamine RNAiMAX Transfection Reagent  following the manufacturer's instructions for 24 h. Cells were then washed with PBS and incubated in DMEM  supplemented with 10% of Lipoprotein Depleted Fetal Bovine Serum  overnight before treatment with 100 μM cholesterol and 10 μg/ml 58035 for 24 h. Cells were then washed with PBS, and the nuclei were stained with DAPI . Cell number was counted in three randomly selected areas for each sample under the microscope .2 incubator for 1 h. Then, 20 μl of 100 μM oligomycin, 22 μl of 100 μl 100 μM FCCP, and 25 μl of 50 μM Rotenone/AA was added to the port A, B, C of the sensor cartridge, respectively. For the cell culture microplate, the medium was replaced by Agilent Seahorse XF DMEM Medium  supplemented with 1 mM pyruvate , 2 mM glutamine , and 10 mM glucose , followed by 1 h incubation in a 37 °C non-CO2 incubator. An XF cell mito stress-test template was used to measure oxygen consumption rate of cells. The data was normalized to protein concentration in Wave 2.6.1 software.The Seahorse assay was conducted using Agilent Seahorse XF Cell Mito Stress Test Kit  and Seahorse XFe96 Analyzer following the manufacturer's instructions. Briefly, 10,000 cells were seeded onto the Seahorse XF Cell Culture Microplate  two days before assaying. One day before the assay, cells were treated with cholesterol and 58035 for 24 h while the microplate cartridge was hydrated by 200 μl Molecular Biology Grade Water  overnight at 37 °C. At the day of assay, the water in microplate cartridge was replaced by prewarmed XF Calibrant  followed by incubation in 37 °C non-COData analyses were performed with GraphPad Prism 8.0.2 software. Data with two groups and six groups were analyzed by unpaired two-tailed student's t test and one-way ANOVA, respectively. Total cholesterol and FC were analyzed using two-way ANOVA. p-values < 0.05 were considered statistically significant.The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


In addition, 8A9 shows cross-reactivity with rhesus lymphocryptovirus (rhLCV) gB. Competitive binding experiments demonstrate that 8A9 and 8C12 recognize novel epitopes that are different from the AMMO5 epitope. The epitopes of 8A9 and 8C12 are mapped to gB D-II, and the AMMO5 epitope is located precisely at gB aa 410–419. We find that 8A9 and 8C12 significantly inhibit gB-derived membrane fusion using a virus-free fusion assay. In summary, this study identifies two gB-specific NAbs that potently block EBV infection of B cells. Our work highlights the importance of gB D-II as a predominant neutralizing epitope, and aids in the rational design of therapeutics or vaccines based on gB.The Epstein–Barr virus (EBV) is the first reported oncogenic herpesvirus that establishes persistent infection in B lymphocytes in 95% of adults worldwide. Glycoprotein B (gB) plays a predominant role in the fusion of the viral envelope with the host cell membrane. Hence, it is of great significance to isolate gB-specific fusion-inhibiting neutralizing antibodies (NAbs). AMMO5 is the only gB NAb but fails to antagonize B-cell infection. It is essential to isolate potent NAbs that can completely block EBV infection of B cells. Using hybridoma technology and neutralization assay, we isolate two gB NAbs 8A9 and 8C12 that are capable of completely neutralizing B-cell infection  The Epstein–Barr virus (EBV) is a member of the γ-herpesvirus family, which establishes a lifelong infection in approximately 95% of human adults worldwide . As the Similar to the other herpesviruses, EBV infection is a complex multistep process that requires the involvement of multiple viral glycoproteins . TypicalEBV gB is the product of the BALF4 open reading frame (ORF) and categorized as a class III viral membrane fusion protein –32. LikeIn this study, we isolated two murine NAbs, 8A9 and 8C12, from gB-immunized mice by hybridoma technology. Both 8A9 and 8C12 showed better neutralizing capacity than AMMO5 by completely neutralizing B-cell infection. 8A9 and 8C12 recognized novel epitopes located on D-II of gB, which was distinct from that of AMMO5. 8A9 and 8C12 exhibited different cross-reactivities with rhLCV gB even though they showed epitope competition. We identified the precise epitope and key residues recognized by AMMO5 for the first time. 8A9, 8C12, and AMMO5 could significantly inhibit gB-mediated membrane fusion. The characterization of 8A9, 8C12, and AMMO5 reveals the key role of the stem region, especially D-II, of gB in eliciting NAbs.2. MDCK cells and 293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). EBV-negative Akata cells, Sp2/0 cells, and hybridoma cells were grown in RPMI 1640 supplemented with 10% FBS. 293F cells were cultured in OPM-293 CD05 Medium (OPM Biosciences).All cell lines were cultured and routinely maintained at 37°C in humidified air containing 5% COO-tetradecanoylphorbol 13-acetate (TPA) (20 ng/ml) and sodium butyrate (2.5 mM) for 12 h and then the medium was replaced by RPMI 1640 with 10% FBS. After 72 h, the cultures were clarified by centrifugation at 1,000 g for 5 min and then the supernatant was filtrated through 0.45-μm filters. Viruses were concentrated 100-fold by centrifugation at 50,000 g for 2.5 h, and the virus pellets were resuspended by RPMI 1640 without FBS. The viruses were stored at −80°C for use.CNE2-EBV-GFP cells were induced by 12-112WY113 and 193WLIW196 replaced by HSV-1 residues 177HR178 and 258RVEA261, respectively. The coding sequence of the rhLCV gB ectodomain (residues 23–685) (UniProt ID: Q8UZD5) was synthesized with residues 112WY113 and 193WILW196 replaced in the same way as for EBV gB. Both gB genes were cloned into pCDNA3.1 with a 6 × His tag at the C-terminus. The plasmids coding for gB domains were constructed by inserting the appropriate DNA fragment into the vectors pCDNA3.1 and pTO-T7-HBc149 (The coding sequence of the EBV gB ectodomain (residues 23–683) (UniProt ID: P03188) was cloned with residues 600 = 0.8 at 25°C for 6–8 h. The bacteria were collected by centrifugating at 7,000 g for 15 min and resuspended by PBS. Then, the supernatant was collected after ultrasonication and centrifugation. The supernatant was heated at 65°C for 30 min and centrifugated at 12,500 rpm for 10 min. The supernatant was further purified by gel filtration using Superdex 200 10/300 GL (Cytiva).Plasmids encoding gB of EBV and rhLCV, and EBV gB D-I to D-V, were transfected into 293F cells. The supernatant was collected and purified with Ni-NTA resin (Cytiva). Plasmids encoding truncated forms of gB domains were transformed into BL21(DE3) competent bacteria. Protein production was induced by adding isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM when ODvia limiting dilution, and purified from mouse ascites using protein A affinity chromatography (Cytiva).Eight-week-old female BALB/c mice were immunized with gB (100 μg/mouse) emulsified with Freund’s adjuvant subcutaneously 3 times at 2-week intervals. Two weeks after the final immunization, mice were boosted again. Three days later, spleen cells of immunized mice were collected and fused with mouse myeloma Sp2/0 cells. The hybridomas secreting gB-specific MAbs were sequentially screened by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. The hybridomas were cloned three times 293F cells were transfected with plasmids encoding heavy and light chains of AMMO5 . AMMO5 w2SO4 solution. Absorbance was measured at 450 nm using a PHOmo microplate reader (Autobio).To assess the binding ability of antibodies or sera, proteins were coated on 96-well ELISA plates with 100 ng/well at 37°C for 2 h. The plates were washed by PBS containing 0.1% v/v Tween-20 (PBST) once and blocked with PBS containing 2% w/v non-fat dry milk (blocking solution) at 37°C for 2 h. The 2-fold serially diluted antibody was added to the wells and incubated at 37°C for 30 min. After washing five times with PBST, 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (GAM-HRP) buffer or goat anti-human IgG (GAH-HRP) buffer was added to each well and incubated at 37°C for 30 min. After washing five times with PBST, 100 µl of tetramethylbenzidine (TMB) substrate (Wantai BioPharm) was added and incubated at 37°C in the dark for 15 min. The reaction was stopped with a 2 M H2SO4 solution. Absorbance was measured at 450 nm using a PHOmo microplate reader.To evaluate antibody binding activities with peptides, streptavidin-immobilized plates (Wantai BioPharm) were coated with biotinylated peptides (200 ng/well) at 37°C for 1 h. Antibody at 1 μg/ml was added to the wells and incubated at 37°C for 30 min. After five washes with PBST, 100 µl of GAM-HRP IgG buffer or GAH-HRP buffer was added to each well and incubated at 37°C for 30 min. After five washes, 100 µl of TMB substrate (Wantai BioPharm) was added at 37°C in the dark for 15 min. The reaction was stopped with a 2 M Hvia covalent coupling. A 70-µl solution of a 1:1 (v/v) of N-ethyl-N-(3-diethylarninopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was mixed to activate the carboxyl groups on the dextran surface. EBV gB was diluted with 10 mM sodium acetate (pH 5.0) to a final concentration of 1.5 µg/ml for coupling. rhLCV gB was diluted with 10 mM sodium acetate (pH 5.0) to a final concentration of 2.5 µg/ml for coupling. In each channel, only flow cell 2 was coated with ligand while flow cell 1 was empty and blocked by ethanolamine as a control. After chip loading, the serially diluted antibody was injected at 30 µl/min for 300 s (association phase), followed by dissociating at 30 µl/min for 600 s (dissociation phase). The results were analyzed by BIAcore Insight Evaluation software, and the curve fitting was performed using a 1:1 binding model.BIAcore 8k (Cytiva) was used for evaluating the equilibrium dissociation constant  of the antibodies. The sensor chip CM5 was coated with proteins To assess the epitope competition with 8A9, EBV gB was coated in sensor chip CM5 at 1.5 µg/ml. 8A9 was diluted to 800 nM and injected at 30 µl/min for 300 s, and antibodies  at 800 nM were injected for 300 s. The epitope competition with AMMO5 was analyzed in the same way.The protein samples mixed with loading buffer containing β-mercaptoethanol were heated at 100°C for 10 min. Then, the mixture was loaded into 4%–12% SurePAGE gels (Gen Script) and run at 180 V for 30 min. Proteins were visualized by Coomassie Brilliant Blue staining for 30 min and destained until the background became transparent.Proteins on the gels were transferred onto polyvinylidene fluoride membranes (Millipore) after SDS-PAGE. The membranes were incubated with a blocking buffer (Wantai BioPharm) at room temperature for 1 h. The membranes were incubated with 8A9, 8C12, and AMMO5, respectively. After five washes with PBST, the membrane was incubated with GAM-HRP or GAH-HRP at room temperature for 30 min. After five washes with PBST, the WesternBright ECL (Advansta) was used for color development.PNGase F (NEB) was used to release the N-linked glycans. PNGase F (10 µl) was added with 100 µg of protein, and the final sample was prepared as 100 µl. The sample was incubated in a water bath at 37°C for 12 h. The deglycosylated proteins were analyzed using SDS-PAGE.The plasmid encoding full-length EBV gB or rhLCV gB was transfected into MDCK cells for 48 h. The transfected cells were seeded on 96-well plates overnight, and then fixed with 4% paraformaldehyde at room temperature for 15 min and washed twice. The cells were incubated with blocking solution (2% BSA in PBS) at 37°C for 2 h and washed with PBST. Antibodies were used for staining by incubation with cells at 4°C overnight. After two washes with PBST, the cells were labeled with donkey anti-mouse AlexaFluor 488 (Molecular Probes) and goat anti-human IgG AlexaFluor 488 (Molecular Probes) at room temperature for 30 min. After two washes with PBST, the cells were stained with DAPI (Invitrogen) for 5 min. The samples were visualized with the Opera Phenix Plus High-Content Screening System (PerkinElmer).6 cells per test. The cells were stained with antibodies at 4°C for 30 min. After two washes, 293T cells were incubated with goat anti-mouse IgG BV421 (BioLegend) and anti-human IgG BV421 (BD Biosciences) at 4°C for 30 min in the dark. Stained cells were analyzed by flow cytometry (FCM) on an LSRFortessaX-20 instrument (BD Biosciences) and the data were evaluated using FlowJo software X 10.0.7 (BD Biosciences).293T cells were transfected with plasmid encoding the full-length rhLCV gB for 48 h. The transfected cells were trypsinized and resuspended by PBS and prepared with 10The gB proteins were analyzed by negative staining electron microscopy as previously reported . Briefly4 Akata cells/well in 96-well plates. The numbers of GFP-positive cells were measured by the LSRFortessaX-20 instrument (BD Biosciences) after incubation with RPMI 1640 containing 10% FBS for 48 h to determine infection rate. Uninfected cells were used as negative control and Akata cells infected with EBV were regarded as infected control. The neutralizing efficiency of antibodies was calculated by the following equation: neutralization efficiency % = [(% of GFP-positive cells in infected control) − (% of GFP-positive cells in the antibody-treated group)]/% GFP-positive cells in infected control.For neutralization of B cells, serially diluted antibodies were incubated with 20 µl of CNE2-EBV-GFP at 37°C for 2 h. The mixture was added into 10To evaluate the purity of gB protein, EBV gB and rhLCV gB proteins were analyzed by an 1120 Compact LC HPLC system (Agilent Technologies) and separated by a TSKgel G3000PW XL column (TOSOH), which was pre-equilibrated with PBS. The flow rate was 0.5 ml/min and protein signal detection was of 280 nm.6 cells/dish. When reaching 80% confluence, the cells were transfected with 2.5 μg each of pCAGGS-gB, pCAGGS-gH, pCAGGS-gL, and pCAG-T7. 293T cells in another dish were transfected by 10 μg of pT7EMCLuc. Twenty-four hours after transfection, 2×105 cells transfected with plasmids encoding gB, gH, gL, and T7 polymerase were trypsinized and incubated with 0.8 μg of antibodies at 37°C for 30 min. Then, the mixture of cells and antibodies was added with 2×105 cells transfected with pT7EMCLuc in a 24-well plate and cultured for 24 h at 37°C in DMEM with 10% FBS. Cells were lysed and luciferase activity was quantified using Dual-Glo luciferase assay (Promega).To evaluate the cell fusion blocking efficiency of NAbs 8A9, 8C12, and AMMO5, 293T cells were inoculated in 10-cm dishes in DMEM with 10% FBS at a density of 2×10p-values were generated by a one-way ANOVA analysis. Data were considered statistically significant at *p < 0.05.All statistical analyses were performed using GraphPad Prism. 50) value of 8.45, 111.30, and 25.99 ng/ml, respectively  assay. 8A9, 8C12, and previously reported NAb AMMO5  showed sectively  did not Ka] of 2.10 × 104 M−1 s−1) and slow-off (dissociation rate constant [Kd] of 2.12 × 10-6 s−1) binding kinetics, resulting in an equilibrium dissociation constant (KD) of 0.10 nM  assay. We found that the three MAbs exhibited specific staining with native full-length gB displayed on the surface of gB-transfected cells, while the negative control antibodies 72A1 and VRC01 had no such effect (Previous reports demonstrated that some MAbs to gB from the herpesvirus family (HCMV and HSV) recognized linear and continuous epitopes , 53. We AMMO5 was the only published NAb targeting gB, while its neutralizing epitope on gB had not been reported . To eval50) value of 0.75 μg/ml, compared to that of 8C12 (5.39 μg/ml) and 72A1 (7.83 μg/ml). 8A9 and 8C12 were the first reported gB NAbs that completely neutralized B-cell infection. Collectively, we had generated two gB antibodies, 8A9 and 8C12, which showed a specific binding ability against recombinant gB and native gB. 8A9 and 8C12 recognized novel epitopes that were different from that of AMMO5. Both 8A9 and 8C12 were capable of neutralizing B-cell infection completely.Next, we evaluated the neutralizing ability of 8A9 and 8C12 in a B-cell infection assay. For comparison, we also included gp350 murine NAb 72A1 and gB NAb AMMO5. VRC01 was used as a negative control. Consistent with a previous report , AMMO5 c112WY113 and 193WILW196) are also hydrophobic and almost identical to that of EBV gB (112WY113 and 193WLIW196) (177HR178 and 259RVEA261). The ectodomain variant of rhLCV gB was constructed and successfully expressed using 293F cells. rhLCV gB contains a furin protease cleavage recognition sequence (RRRRR) that is identical to EBV gB , share a high degree of sequence identity , 56. rhL50 = 412.00 ng/ml) was ~50-fold weaker than that against EBV gB (EC50 = 8.45 ng/ml). The binding affinity of 8A9 with rhLCV gB (KD = 106.00 nM) (KD = 0.101 nM)  (AMMO5 showed strong reaction with P39 (RGST422)   were included in P6 (73TRENHTEGLLMVFKDNIIPY92) of the overlapping peptide library . Alanine mutations to Pro413, Pro415, and Arg419 resulted in diminished binding activity with 1–2 orders of magnitude decrease in EC50 values   and deglycosylated gB . The data suggested that the epitopes of 8A9, 8C12, and AMMO5 were not glycosylation-dependent.The glycosylation of viral glycoproteins plays an essential role in mediating protein folding, interaction with host receptor, and immune evasion . EBV gB EBV mainly infects epithelial cells and B cells by different routes . The proin vivo. Most animal models for EBV infection are mainly based on infection of B lymphocytes, and associated with the formation of lymphomas  , while win vitro firstly. Moreover, the construction of a humanized 8A9 is required to avoid human anti-murine antibody (HAMA) response. Affinity maturation is also needed since 8A9 only showed modest binding affinity with rhLCV gB  can be found in the article/The animal study was reviewed and approved by the Ethics Committee of Xiamen University Laboratory Animal Center.JH, NX, XZ, and YC designed the studies. JH, DW, LZ, QW, KC, WZ, and YY performed experiments. JH, DW, LZ, QW, and JC analyzed the data. JH, XZ, and YC wrote the paper. All authors contributed to the article and approved the submitted version.The study was supported by grants from the National Natural Science Foundation of China (grant 82073756) and XMU Training Program of Innovation and Enterpreneurship for Undergraduates.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.


COL4A3 gene, which is considered disease-causing. All patients were from the continental or island part of Croatia. Clinical, laboratory, and histopathological data collected from the medical records were analyzed and compared to understand the clinical course and prognosis of the affected patients. At the time of biopsy or first clinical evaluation, the mean age was 31 years . Hematuria was present in 33 patients (97.1%) and 19 (55.9%) patients had proteinuria. There were 6 (17.6%) patients with hearing loss, 4 (11.8%) with ocular lesions, and 11 (32.4%) with hypertension. Twenty-three (67.6%) patients had proteinuria at follow-up, and five (14.7%) patients with the median age of 48 years (range: 27–55) progressed to kidney failure, started dialysis, or underwent kidney transplantation. Of the 13 patients who underwent kidney biopsy, 4 (30.8%) developed focal segmental glomerulosclerosis (FSGS), and 8 (66.7%) showed lamellation of the GBM, including all patients with FSGS. It is essential to conduct a detailed analysis of each collagen type IV genetic variant to optimize the prognosis and therapeutic approach for the affected patients.Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are part of the spectrum of kidney disorders caused by pathogenic variants in α3, α4, or α5 chains of the collagen type IV, the major structural component of the glomerular basement membrane (GBM). Using targeted next-generation sequencing (NGS), 34 AS/TBMN patients  from 12 unrelated families were found positive for heterozygous c.2881+1G>A variant of the  COL4A3, COL4A4, and COL4A5 genes which encode α3, α4, or α5 polypeptide chains of collagen type IV (COL4), the main component of the human basement membrane in glomeruli, inner ear, and the eye [Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are inherited renal disorders characterized by a structural defect of the glomerular basement membrane (GBM). The defect is caused by variants in the  the eye .COL4A5 gene (encoding α5 chain) located on the X chromosome, thus causing the X-linked type of AS (XAS). Autosomal recessive AS (ARAS) or autosomal dominant type of AS (ADAS) are caused by variants in the COL4A3 or COL4A4 genes  [COL4A3 and/or COL4A4 variants in about one in 100 [The prevalence of AS is 1:5000 [ctively) . Analysie in 100 . XAS, ARIn most cases, AS clinically presents with hematuria. Other characteristics are ultrastructural changes of GBM, sensorineural hearing loss, and ocular lesions . In clasCOL4A3, COL4A4, and COL4A5 pathogenic variants in the Genome Aggregation Database  showed that the heterozygous COL4A3 or COL4A4 variants are present in about 1 in 100 individuals, making them the most frequent genetic kidney disorder [COL4A3 and COL4A4 patients are usually diagnosed with TBMN. Its primary clinical feature is persistent microscopic hematuria [COL4A3 and COL4A4 variants and benign familial hematuria has been observed in several studies [COL4A3 and COL4A4 variants had a benign course, with some individuals presenting with progressive hematuria and proteinuria, FSGS, hypertension, and KF [COL4 variant can have different clinical and histological findings, even in the same family [COL4 gene variants. This term was discussed at the latest International Workshops on Alport Syndrome but is still under consideration by the wider community [Analysis of disorder . Based oematuria , and theematuria . A corre studies . However, and KF , 32–39. e family . For theommunity .COL4 genes, with the location and type of variant largely determining the clinical course and disease prognosis [COL4A3 gene identified in 12 families from the continental or island part of Croatia.For the past 20 years, AS and TBMN research has been focused on the correlation of genotype and phenotype, describing the entire spectrum of phenotypes caused by variants in the rognosis , 41, 42.COL4A3 c.2881+1G>A was detected in 39 participants from 12 unrelated families. Five patients declined to participate in the nephrological assessment and subsequent follow-up. Thus, they were excluded from the study. Out of 34 patients, 13 underwent kidney biopsy.This study was conducted as a part of the research project “Genotype–phenotype correlation in Alport’s syndrome and thin glomerular basement membrane nephropathy” funded by the Croatian Science Foundation (IP-2014-09-2151), approved by the Ethics Committee of the University of Zagreb School of Medicine . All participants signed informed consent before blood sample collection and genetic testing. After genetic counseling, urinalysis, family members’ recruitment, and blood sampling for targeted next-generation sequencing (NGS), the heterozygous variant https://www.mdcalc.com/ckd-epi-equations-glomerular-filtration-rate-gfr) for adults and the Bedside Schwartz equation for children [2) with urinary abnormality (hematuria and proteinuria), stage 2 mildly decreased renal function (eGFR 60–89 ml/min/1.72 m2), stage 3a mildly to moderately decreased renal function (eGFR 45–59 ml/min/1.72 m2), stage 3b moderately to severely decreased renal function (eGFR 30–44 ml/min/1.72 m2), stage 4 severely decreased renal function (eGFR = 15–29 ml/min/1.72 m2), and stage 5 (eGFR < 15 ml/min/1.72 m2) denotes KF. A correlation of clinical and histopathological data and a genealogy study was also performed.The patient demographic and clinical data  stage, beginning of dialysis, transplantation, and follow-up data) were collected from medical records. We considered hematuria to be a finding of more than five red blood cells per high-power field (area visible under the microscope at 400× magnification). Proteinuria was defined as a positive result in the urine dipstick test or proteinuria higher than 150 mg in 24-h urine. The estimated glomerular filtration rate (eGFR) values were calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation , immunofluorescent analysis , and EM. The stages of interstitial fibrosis and tubular atrophy (IFTA) were defined as mild (stage 1), affecting less than 25% of the specimen; moderate (stage 2), affecting 25%–50%; and severe (stage 3), affecting more than 50% of the specimen. In all participants (except one patient without glomeruli in tissue prepared for EM), the GBM thickness was measured on EM images using iTEM software (Olympus Soft Imaging Solutions GmbH). Altogether three measurements of the GBM thickness per capillary loop on ten random capillary loops per specimen were made  using a modified direct method of measuring GBM thickness as previously described by Haas .The genomic DNA was isolated from peripheral blood samples using Zymo Quick-DNA Miniprep Plus Kit , following the manufacturer’s procedure. DNA concentration was measured using a High Sensitivity Qubit quantification kit .COL4A3, COL4A4, and COL4A5 genes. A 20 ng of genomic DNA was used to amplify the targeted genes in a single-tube multiplex reaction. Adapter sequences and molecular indexes were incorporated during PCR steps, and amplicon libraries were purified using magnetic beads Agencourt AMPure XP reagent . Library quality was assessed with Agilent Bioanalyzer HS DNA Kit , showing adequate sample sizes and concentrations. Libraries were then quantified using a High Sensitivity Qubit quantification kit  and pooled to generate a sequencing library with a final loading concentration of 50 pM.Libraries with amplicon sizes ranging from 125 to 175 base pairs (bp) were prepared following the manufacturer’s procedure using AmpliSeq Library Plus and AmpliSeq Custom DNA Panel , which includes two primer pools with 89 and 87 primer pairs, respectively, that can amplify all coding and flanking splice regions of Subsequent Illumina sequencing was carried out on the Illumina iSeq 100 System platform  with Standard flow cell following the manufacturer’s instructions. The mean coverage depth of all amplicons achieved by targeted NGS for all samples was 270×. Sequencing generated the corresponding FASTQ files, and bioinformatical analysis was performed using the Illumina VariantStudio software  .COL4A3 at genomic position chr2: 228149062 (variant described according to reference genome GRCh37) was confirmed with standard dye-terminator sequencing. Genomic DNA was amplified using forward (FW) 5’ GGG GGA ACA AGG AGA TAA AGG A3’ and reverse (RV) 5’ AAA CAC TGG CCC TCA CTG TC3’ primers and EmeraldAmp MaX HS PCR Master Mix . PCR products were purified using ExoSAP-ITTM PCR Product Cleanup Reagent . Sanger sequencing was performed on the ABI310  with BigDye v1.1 chemistry . Results were visualized with Vector NTI software .The splice donor variant c.2881+1G>A found in COL4A3 variant c.2881+1G>A (LRG_230t1:c.2881 +1G>A) located at the genomic position chr2:228149062 (GRCh37.p13) affects the splice-donor sequence of intron 34 of COL4A3 gene potentially leading to exon skipping. We detected no other variants in COL4A3, COL4A4, or COL4A5 genes. However, we cannot exclude additional genetic and nongenetic factors which may contribute to disease severity. The c.2881+1G>A variant was not found in the GnomAD database (https://gnomad.broadinstitute.org). However, the variant was reported in two patients  from Germany in Leiden Open Variant Database (LOVD) and described as pathogenic, causing ADAS (https://databases.lovd.nl/shared/variants/in_gene?search_geneid=%3D%22COL4A3%22&search_VariantOnTranscript/DNA=%3D%22c.2881%2B1G%3EA%22). There were no additional clinical or histopathological data in those reports. Furthermore, in silico bioinformatics tools, VarSEAK (https://varseak.bio/) and Mutation Taster (http://www.mutationtaster.org) predicted disease-causing effects. Nevertheless, the expected damaging effect of c.2881+1G>A should be confirmed with functional studies.The described COL4A3 gene at the same position but with a different nucleotide substitution and classified it as pathogenic (https://clinvarminer.genetics.utah.edu/submissions-by-variant/NM000091.4%28COL4A3%29%3Ac.2881%2B1G%3ET).The Gharavi Laboratory at Columbia University reported variant c.2881+1G>T in the COL4A3 c.2881+1G>A variant could be considered a founder variant as it was detected with high frequency in a homogeneous and closed community on the island Pašman, Croatia. Guidelines of joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [According to our knowledge, athology  and Recoathology  were folt-test, Mann–Whitney U test, and Kruskal–Wallis test, when appropriate. The occurrence of adverse events  was calculated utilizing the Kaplan–Meier method and compared between groups with the log-rank test. The hazard ratio (HR) for adverse events was estimated by Cox regression analysis. As Alport spectrum disorders are genetic disorders and disease onset data retrieved during patients’ interviews are subjective, we decided to mark the starting age in the analysis of renal survival time  as 0 years. All data were analyzed using IBM SPSS Statistics for Windows v.29.0 . All statistical tests were two-sided, and intergroup differences with P < 0.05 were considered significant.Descriptive statistical analysis methods were used to evaluate demographic and clinical data. Mean, median, standard deviation (SD), and interquartile range (IQR) were calculated for numeric variables, when appropriate. Frequency values were used to describe categorical data. Statistical analyses of data were performed using Fisher’s exact test, unpaired independent-samples COL4A3 c.2881+1G>A at genomic position chr2:228149062 , associated with ADAS and ARAS (PVS1). The variant was not found in GnomAD exomes (coverage 61.8%) nor GnomAD genomes (coverage 30.3%) despite good coverage (PM2). The variant has a pathogenic computational verdict based on four pathogenic predictions from DANN, EIGEN, FATHMM-MKL, and MutationTaster versus no benign predictions. In silico software results were DANN score: 0.9936; EIGEN prediction: Pathogenic ; FATHMM-MKL prediction: Damaging (coding score 0.989); MutationTaster prediction: Disease-causing (probability 1). The comparison of an in-house database of 50 healthy individuals tested for variants in COL4A3, COL4A4, and COL4A5 genes also support our analysis results. We suspect COL4A3 c.2881+1G>A to be a founder variant as it was detected with high frequency in a homogeneous and closed community on the Pašman island in Croatia. It is also a pathogenic variant according to in silico analysis and the fact that it is located in the non-coding splice-site locus.Atotal of 39 participants from 12 unrelated families reported positive for heterozygous variant 28149062 . There wCOL4A3 c.2881+1G>A variant, are shown in COL4A3 c.2881+1G>A variant and was also positive. The hearing loss was detected in 6 (17.6%) patients, with 4 of them being members of the same family . Ocular abnormalities were detected in 4 (11.8%) patients, whereas 11 (32.4%) patients presented with hypertension. The mean eGFR at the time of biopsy or first clinical evaluation was 85.9 ml/min/1.72 m2 . According to eGFR, 20 (58.8%) patients had stage 1 CKD, 8 (23.5%) had stage 2, 3 (8.8%) had stage 3b, 2 (5.9%) had stage 4, and 1 (2.9%) patient (D11) had stage 5 CKD  clinical features of 34 out of 39 positively tested patients , with proven ge 5 CKD . One  at the follow-up was 48 years (IQR: 41.3–54.3 years), with the youngest patient having 27 and the oldest 55 years. Hematuria was present in 30 patients (88.2%), whereas 6 (17.6%) had macrohematuria. Follow-up data also showed that four more patients developed proteinuria . The mean eGFR on follow-up was 78.9 ml/min/1.72 m2 . Furthermore, according to calculated eGFR values, 17 (50%) patients had stage 1 CKD, 9 (26.5%) had stage 2 CKD, 2 (5.9%) had stage 3b, 4 (11.8%) had stage 4, and 2 (5.9%) patients had stage 5 CKD , as shown in ge 5 CKD .P = 0.012) between patients without proteinuria , where none had deterioration of the CKD stage, and patients with proteinuria , where 9 (26.5%) showed a deterioration of the CKD stage (By comparison with baseline patients’ characteristics in 24 patients (70.6%) at follow-up, the CKD stage was the same; only 1 (2.9%) patient experienced an improvement, whereas the CKD stage worsened in 9 (26.5%) patients. There is a statistically significant difference .Although the Kaplan–Meier method and the log-rank statistics for time to an adverse event  showed no statistically significant association with proteinuria , Cox regKidney biopsy data were available for 13 patients . HistopaAs there were no glomeruli in one specimen for EM, the ultrastructural analysis was conducted for 12 patients. The average GBM thickness was 204.7 nm . Lamellation was present in eight (66.7%) cases; in six (50.0%) was only focally present .COL4A3 c.2881+1G>A detected with NGS. After collecting clinical and histopathological data from the participants and obtaining the NGS results, we performed data correlation and genealogy study, generating family pedigrees  variants were also identified in patients with familial FSGS, broadening the spectrum of structural disorders of the GBM [COL4 variants, has been considered a benign disorder with an excellent prognosis, several publications convincingly show that this is inaccurate [ the GBM , 52–54. accurate , 9, 33. accurate .COL4 variants, with clinically distinct but similar ultrastructural manifestations, were better understood as a spectrum of AS. This would cover all disorders from benign familial hematuria  to classical AS with undetectable COL4 in the GBM, eventual progression to KF, auditory and visual disturbances, and everything in between these two extremes. Given that a variant that causes hematuria in one person can cause progressive kidney disease in another, Miner [COL4A3, COL4A4, or COL4A5 genes.In his research in 2014, Miner [r, Miner , the terCOL4A3 and COL4A4 variants, Matthaiou et al. [COL4A3 c.2881+1G>A showed that at the time of biopsy or first clinical evaluation, hematuria was present in 97.1%, proteinuria in 55.9%, hearing loss in 17.6%, and ocular abnormalities in 11.8%, whereas at the time of the last follow-up, 30 (88.2%) patients had hematuria, 23 (67.6%) had proteinuria, and 5 (14.7%) developed KF, started dialysis, or underwent kidney transplantation by the median age of 48 years (age range 27–55 years). Even though we would expect all patients to have hematuria, three of them had hematuria at the time of biopsy or first clinical evaluation and no hematuria on follow-up while one patient had no hematuria at all. These facts highlight the importance of regular follow-up due to intermittent hematuria which is described in patients with TBMN [In 2020, in their systematic literature review of heterozygous u et al.  presenteu et al. . On lighu et al. . In compith TBMN . FSGS waCOL4 variants has been expanded to include disorders from isolated hematuria due to TBMN to more severe phenotypes, such as ARAS, ADAS, and FSGS, which can progress to KF. One hypothesis is that the spectrum of disorders caused by COL4 variants is a multifactorial disorder whose phenotypic expression depends on the interaction of pathogenic variants affecting primary genes, modifying genes, and environmental factors [The phenotypic spectrum of  factors , 37, 49.COL4 genes, such as deep intronic variants that may contribute to disease severity, cannot be detected with NGS [However, there are two limitations of our study considering the additional molecular analysis. For now, we are unable to conduct the sequencing of other genes associated with hematuric nephritis and genetic FSGS and to analyze additional genetic modifiers that aggravate severe phenotypes. Also, some variants in with NGS .COL4A3 c.2881+1G>A is disease-causing. These patients differ not only in clinical presentation but also in histopathological findings. It is characterized by hematuria and expressed extrarenal symptoms in 20.6% of patients and potential risk of developing FSGS and progression to KF in 14.7% of affected individuals. Proteinuria should be considered as an indicator of a potential decline in renal function since 26.5% of patients with proteinuria showed a decline in renal function whereas all patients without proteinuria had maintained renal function between the time of kidney biopsy and last follow-up.The suspected founder variant COL4A3, COL4A4, and COL4A5 variants should always be considered potentially serious and, therefore, a detailed and constructed clinical, histopathological, and genetic assessment of each patient should be performed. An appropriate therapeutic and prognostic approach should be adjusted for each individual according to the severity of the clinical course, morphological changes, and type of variant detected with genetic analysis.Alport spectrum disorders caused by Conflicts of interest: Authors declare no conflicts of interest.Funding: This research was conducted as part of the research project “Genotype-phenotype correlation in Alport’s syndrome and thin glomerular basement membrane nephropathy,” funded by the Croatian Science Foundation (IP-2014-09-2151).