We demonstratethe cumulative effect of solvent and additive on delivering multipleperformance benefits and safety of the battery. The faster diffusionrate of K + solvation shell decreases the rate of PC decomposition,thereby reducing its cointercalation. Additionally, the optimum concentrationof KPF6, i.e., 0.1 M constructs a robust and insolubleLiF-rich electrode/electrolyte interphase, further suppressing graphiteexfoliation and Li dendrite formation. The stable cyclability is achievedby enhanced Li + transportation through the LiF-rich interphase,enabling an exfoliation-free and dendrite-free graphite anode in theternary electrolyte.Propylene carbonate (PC) is a promising solvent for extendingtheoperating temperature range for lithium-ion batteries (LIBs) becauseof its high dielectric constant and wide temperature range stability.However, PC can cause graphite exfoliation through cointercalation,leading to electrolyte decomposition and subsequent irreversible capacityloss. This work reports the formulation of a ternary electrolyte withthe introduction of an inorganic salt additive, potassium hexafluorophosphate(KPF One of these key factors is the operating temperatureof LIBs, which is limited by the temperature range of the electrolyte(stability window), affecting its inherent properties such as ionicconductivity, viscosity, and charge-transfer resistance.4 Therefore, in order to expand the operating temperature range ofa LIB, the electrolyte’s temperature range is often modifiedby incorporating solvents and/or additives. One of the electrolytesolvents, propylene carbonate (PC) has a wide temperature range, i.e.,−48.8 to 242 °C, which makes it a desirable organic solventfor more extreme temperature operations.6 However, theexfoliation of graphite layers caused by PC cointercalation resultsin electrolyte decomposition, which gives rise to concerns for present-dayLIB.10Lithium-ion batteries(LIBs) have achieved great success in portableelectronics and are an established technology for energy storage systemssince their original commercialization in 1991.+ ion transportationbetween the electrodes in conventional LIBs. Usually, a mixture ofcyclic carbonates is combined with linear carbonates in various proportionsto produce an electrolyte solvent with suitable viscosity and ionicconductivity. Although the high dielectric constants of cyclic carbonates(ethylene carbonate (EC) and PC) promote dissolution and dissociationof Li salt, the high viscosity leads to their low ionic conductivity.11 Therefore, less viscous linear carbonates (dimethylcarbonate (DMC), ethyl methyl carbonate (EMC), and diethyl carbonate(DEC)) are added to enhance the Li + ionic conductivityof the electrolyte.12 Among all the electrolytesolvents, EC is irreplaceable as it becomes reduced first on the anodesurface14 forming aflexible electrode/electrolyte interphase–solid electrolyteinterphase (SEI).15 However, EC has a highmelting point (36.4 °C), hence staying in the solid state atroom temperature. Therefore, EC is more likely to crystallize evenin the presence of linear carbonate (when temperature decreases),resulting in increased viscosity and charge-transfer resistance. Thisphenomenon is more pronounced especially when the battery is operatedat temperatures closer to subzero °C, thus limiting the workingtemperature range.16 Therefore, one of the approachesis to incorporate PC as a third solvent in order to form a ternaryelectrolyte, which reduces the liquidus temperature , leading to possible improvements in both theionic conductivity and the cyclability of the cell compared to thebinary electrolyte system.19Liquid electrolytes comprising solvents, a Li salt, and additivesact as a medium for reversible Li 8 Furthermore,the high dielectric constant of PC facilitates the dissociation ofsalts in the electrolyte. However, graphite anodes are not compatiblewith PC, as it is unable to stabilize the SEI film on the anode surface.8 Subsequently, PC cointercalates into the graphite layers with Li +, leading to its exfoliation. Additionally, this loosely boundSEI film does not block electrons efficiently, resulting in the reductionof electrolyte and Li +, thereby promoting electrolytedecomposition and Li metal deposition, respectively, even at low chargingrates.15 These phenomena consume active Li throughelectrolyte decomposition, promoting SEI damage and delamination andthereby decreasing the reversible capacity of the cell.PC is a suitable solventfor battery electrolytes as it has structuralsimilarities with EC (extra methyl group attached to ring) in additionto the wide temperature range.20 In this regard, the most effective approach is to introduceadditives into the electrolytes for building an effective SEI filmon graphite anodes with PC-based electrolytes. Recently, it has beendemonstrated that the incorporation of a small amount of salt additivescan suppress PC decomposition on graphite materials by improving theperformance of the cell.24 Similarly, Chandrasiri et al. discovered that bothcesium and potassium acetate result in thinner SEI layers in PC-basedelectrolytes compared to other alkali acetates.22 Numerous studies have been conducted further on salt additivesin PC-rich electrolytes and have been found to have significant performanceenhancement with graphite anodes.24 For instance, the CsPF6 additive suppresses graphiteexfoliation in PC-rich electrolytes by producing an ultrathin andcompact SEI film on its surface.24 In addition,this modified electrolyte provides excellent wide-temperature performancedue to the extended temperature stability of the PC solvent.19 Furthermore, CsPF6 as an electrolyte additive efficientlyinhibits Li dendrite growth enabling improved Li + transportationthrough the SEI layer.28 However, potassium electrolyteadditives have not been thoroughly investigated in PC-containing electrolyteswith regard to impacting both graphite exfoliation and Li dendriteformation, thus not demonstrating the multiple PC solvent-potassiumadditive correlations on graphite. Moreover, the impact of additiveconcentration along with PC solvent has not yet been fully explored.Therefore, this study is conducted to establish the understandingof the electrolyte solvent, additive, and its combined effect on theinhibition of Li dendrite formation and growth and graphite exfoliationin order to enhance the cell’s performance.A stableSEI construction is essential to suppress graphite exfoliationin order to maintain the cyclability of the cell.6), is systematically investigated with PC solventon graphite in order to evaluate the impact of the additive and itsunderlying mechanism. The primary interest of this comprehensive studyis to formulate an optimized electrolyte by investigating the influenceof additive concentrations and volume proportions of PC and EC solventsfor suppressing graphite exfoliation and Li dendrite formation/growth.Ultimately, this will deliver a superior electrochemical performanceand expand the lifetime of the battery.In this work,a potassium additive, potassium hexafluorophosphate(KPF22.10.6Mn0.2Co0.2O2 (Targray NMC 622) electrodes were providedby the CAMP Facility, Argonne National Laboratory, USA. The detailsof the electrodes are stated in Table S4. The commercial electrolyte consisting EC:EMC (3:7 v/v), 1 M lithiumhexafluorophosphate (LiPF6), and 1 wt % VC (vinylene carbonate)named as RD281 was used in this work . Batterygrade EC , PC , and EMC  solvents along with LiPF6  and KPF6 ( ≥ 99 % ) were purchasedfrom Sigma-Aldrich for formulation of electrolytes. Both Li and Ksalts were dried under a vacuum to remove the excess moisture. Theformulation of electrolytes was carried out inside an argon-filledMbraun glovebox (O2 and H2O < 0.5 ppm). Theconcentrations of EMC, LiPF6, and VC additive are keptconstant in order to avoid its influence on the electrochemical performanceof the cell. This implies the volume percentage of PC varies withthe volume percentage EC and vice versa.Single-side-coated artificialgraphite (Hitachi MagE3) and LiNi2.2Graphiteand NMC 622 electrodes were vacuum-dried overnight at 50 °C beforeuse. These electrodes were cut into 15 and 14.8 mm Ø and 15 mmØ, respectively, and assembled into Hohsen 2032-type coin cellsinside the glovebox using a Celgard—2325 separator (19 mm Ø)and modified electrolytes of 100 μL. Graphite | NMC 622 fullcells (N/P ratio = 1.2) cycling was carried out in constant current-constantvoltage (CCCV) charging and constant current (CC) discharging modein 3–4.2 V voltage range. Two formation cycles were employedat C/20 (∼0.14 mA), followed by cycling at C/5 (∼0.55mA) to 100 cycles at room temperature. The electrochemical propertiesare evaluated on a BTS (Battery Technology Source) battery tester.ECC-PAT-core three-electrode cells were used for monitoring thepotential of each electrode distinctively with respect to the referenceelectrode. Graphite and NMC 622 electrodes were cut into 18 mm Ødiscs and assembled into an EL-cell type using NMC 622 as a workingelectrode (WE), graphite as a counter electrode (CE), and inbuiltLi ring as a reference electrode (RE) in the insulation sleeve. Theinsulation sleeve had an inbuilt Whatman Borosilicate glass fiberseparator of 260 μL thickness. EL-cells were cycled in CC modein the 3–4.2 V voltage range.Electrochemical impedancespectroscopy (EIS) was measured usinga three-electrode EL-cell at 50 % SoC (state-of-charge) in ambienttemperature. EIS measurements were taken place after the first cycleand every 10 cycles until 100 cycles. All the measurements were conductedin VMP3 potentiostat (Bio-Logic) in the frequency range of 500 kHz–10mHz with a voltage amplitude of 10 mV. The cells were held for 30min of relaxation time to achieve the equilibrium state prior to EISmeasurements. All of the EIS spectra were fitted with a simplifiedRandles circuit using Zview software.2.3The coincells were disassembled after 100 cycles under fully discharged conditions(3 V) inside the glovebox to reduce moisture contamination. Afterward,an airless transfer device was used to transfer the cycled graphiteanodes from the glovebox to the equipment chamber for postmortem characterization.The cycled electrodes are cleaned carefully with dimethyl carbonate(DMC) to remove the excess electrolyte salt deposits before transferringthe samples to the equipment chamber.Scanning electron microscopy(SEM) was performed to investigate the morphological evolution ofthe cycled graphite anodes. The SEM images were recorded in a ZeissSigma field-emission SEM (FM-SEM)  equipped with anenergy-dispersive X-ray spectrometer (EDX). The images were takenwith an accelerating voltage of 10 kV and an aperture size of 60 μm.In-lens detection was used to collect the SEM images. EDX was performedto determine the elemental composition of the potassium dendrites.29 method using Topas Academic(v6) software.30 Since the measurementswere taken in reflection mode, the samples could be displaced to certaindegrees in the direction normal to the sample plane, which introducedheight error. To address this height error, first, the reflectionfrom the copper current collector was fitted for each sample usinga cubic (Fm3̅m) unit cellwith fixed lattice parameter 3.615 Å,31 with a refinement in sample displacement (SD). This method is reasonable,as the Cu current collector does not participate in the electrochemicalredox reactions. Following this, the d-spacing andpeak width ) of graphite was obtainedby fitting the 002 reflection with a hexagonal (P63/mmc) unit cell, with the refinement in its unitcell parameters (a and c). In this process, the SD obtained from Cu111 reflection was used for fitting graphite 002 reflection (importantto note: SD is not refined again for fitting graphite 002 reflection).In all refinements, a third-degree Chebychev polynomial backgroundfunction was used. The peak shapes were modeled using a pseudo-Voigtfunction combined with an additional function to account for the peakasymmetry .X-ray diffraction (XRD) was conducted in a Malvern PANalyticalEmpyrean diffractometer with nonmonochromatic Cu Kα radiation,using a PiXcel3D detector operating in 1D scanning mode. Data werecollected in Bragg–Brentano (reflection) mode from 5°to 47° (2θ) with 0.013° step size and scan speed of0.011°/s. Afterward, the XRD peaks were analyzed by the Pawleyrefinement5/2 peak, recorded from a polycrystalline Ag sample.XP spectra were recorded at a takeoff angle of 90° and at a passenergy of 20 eV (resolution ∼0.4 eV). In order to negate surfacecharging effects, the surface was flooded with a beam of low-energyelectrons during the experiment. The recorded binding energies wererecalibrated by referencing the C – C/C–H componentof the C 1s spectra to 284.8 eV during fitting using the CasaXPS softwarepackage. The data were fitted using Shirley backgrounds and mixedGaussian–Lorentzian (Voigt) lineshapes.X-rayphotoelectron spectroscopy (XPS) measurements were performedin a Kratos Axis Ultra DLD spectrometer  witha monochromatic Al Kα X-ray (1486.7 eV) source for excitation.The work function of the spectrometer was calibrated using Fermi edgeand 3d–6 mbar were maintained for conducting SIMS experimentsto avoid the collision of ejected secondary ion species with the backgroundgas molecules. Both positive and negative ion mode mass spectra wereobtained by sputtering Ga + source ions on the graphiteanodes at an accelerating voltage of 30 kV and beam current of 0.5nA. MASsoft Professional 7 software was used for recording the massspectra data. SIMS mapping was recorded with ion beam of 10 pA usinga Hiden Analytical SIMS mapper installed in Scios FIB/SEM. The mappingimages were acquired using the acquisition time set to 0.6 ms (dwelltime of 0.4 ms and settle time of 0.2 ms) with 0.01 increment.SIMS experimentswere performed in an FEI Scios dual-beam scanningelectron/focused ion beam (FIB) microscope equipped with a QuadrupoleMass Analyzer (QMA). High vacuum conditions below 5 × 10 33.16 and volume percentages of PC andEC, as listed in Table S1. The formationcycle voltage profile is presented in 6 additive . An unusual slope changein voltage profile at ∼3.37 V (Q/dV vs V) in Figure S1a), delivering a low specificdischarge capacity of ∼88 mA h/g and Coulombic efficiency (CE)of 42.4 %  in Figure S1b) demonstrate that the electrolyte decompositionis diminished in E-10PC compared to E-20PC. This establishes thatthe SEI layer produced on the graphite anode in 10 vol % PC electrolytesis more efficient in restricting electrolyte decomposition comparedto 20 vol % PC-based electrolytes. However, relatively high polarizationis observed in the E-10PC voltage profile, indicating low charge acceptanceof graphite , respectively, with the corresponding reduction peaksduring discharge.32 Similarly, the polarizationis decreased in the E-10PC-0.1 M electrolyte, achieving dischargecapacity of ∼161 mA h/g and CE of ∼82.7 % . The shiftingof the oxidation peaks in the incremental capacity plot (dQ/dV vs V) toward theleft and reduction peak toward the right direction indicates thatthe polarization is reduced with the addition of 0.1 M KPF6 into 10 vol % PC electrolyte. For instance, the first oxidationpeak at ∼3.57 V in E-10PC  shifts to ∼3.5 V with theaddition of 0.1 M KPF6 (green-colored plot), indicatingreduced polarization in E-10PC-0.1 M electrolyte. The reproduciblecycling data and CE are presented in parts S2 and S3.Ultimately, 0.1 M KPF2CO3 throughthe ring opening of PC during the formation cycle. This SEI constituent,e.g., LPDC is less cohesive while depositing on the graphite surface,and more soluble compared to lithium ethylene dicarbonate (LEDC),the decomposition product of EC. It is, therefore, unable to forma solid and compact SEI layer.8 Additionally,propene gas (CH3 – CH=CH2), whichis a byproduct in both schemes 1 and 2 in 10 Nevertheless, the addition of 0.1 M KPF6 in both 10 and 20 vol % PC significantly enhances the capacityof the cell by suppressing electrolyte decomposition (Rs), SEI resistance (RSEI), and charge-transfer resistance (Rct) for E-20PC-0.1 M . Lower PC content produces less LPDC(more LEDC from higher EC) and propene gas and hence declines theprobability of graphite exfoliation through cointercalation. Thisphenomenon produces a relatively stable SEI on the graphite surface,ensuring longer-term cyclability. However, the graphite potentialmeasured from three-electrode cells indicates a developed polarizationin the first cycle , a compactSEI filmis produced on the graphite anode, which suppresses electrolyte decompositionand polarization developed in E-20PC and E-10PC, respectively. Thisdemonstrates the superior performance of the cells cycled with E-20PC-0.1M and E-10PC-0.1 M electrolytes  of graphite isobtained from the 002 reflection and compared to the anode cycledwith commercial electrolyte (RD281) along with a pristine anode forreference. The 002 reflection of the pristine graphite anode presentedin Figure S5 results in an interlayer d-spacing of 3.354 Å in 34 The refinement of graphite cycledwith RD281 yields d-spacing of 3.354 Å and fwhmof 0.046° close to the value obtained from pristine graphite,indicating the intact graphite layers with no exfoliation upon cyclingwith the commercial electrolyte. However, a relatively broad 002 reflectionis seen for E-20PC  in Figure S6 shows the SEM images of graphite exfoliation withregard to different concentrations of KPF6 additive in20 vol % PC electrolytes. Electrolyte decomposition products are alsoidentified on the exfoliated layers, presented as the orange arrowin A probablemechanism of extensive electrolyte decomposition 1g in E-2Figure S5), therefined d-spacing and fwhm are in close proximityto those of pristine graphite, demonstrating no sign of exfoliationin graphite layers. However, Li0  depositionobserved in 6 additive (E-10PC-0.1M) mitigates the dendritic growth as seen in Figure S7 denotes the impact of KPF6 additive concentrations onLi dendrite formation in 10 vol % PC electrolytes.In the case of E-10PC S5, ther3.36 (0.1 M) is found to be the optimum concentrationin 20 and 10 vol % PC electrolytes, which inhibits graphite exfoliationand Li dendrite formation, respectively. However, the electrochemicalperformance suffers as the concentration of KPF6 is increasedto 0.15 M. The specific discharge capacities and CE decline in both20 and 10 vol % PC electrolytes as shown in Q/dV vs V in Figure S8). Morphological investigation in SEM images and the correspondingEDX spectral mapping in + transportation. In addition, these also act as defect sitesfor further Li deposition and subsequent electrolyte decomposition,deteriorating the cell’s performance.0.1 M KPF+ as follows.The morphological investigations of E-20PC-0.1and E-10PC-0.1 Mshow no visible indication of potassium deposition on the graphitesurface. However, for confirmation, the potassium deposition potentialis calculated using the Nernst Equation with respect to Li/Li 36 The calculated potassium deposition potential with respect to theadditive concentration is presented in Table S2. This means that potassium deposition occurs when the graphite anodereaches this potential. Figure S9 confirmsthe maximum potential achieved by graphite is ∼0.08 V in 0.1M KPF6 concentration, which is higher than the K depositionpotential. This ensures that potassium dendrites are absent in 0.1M KPF6 concentration, i.e., in E-20PC-0.1 and E-10PC-0.1M electrolytes. Therefore, potassium remains in the K + form, and its solvation shell directs the SEI formation via enhanceddiffusion through E-20PC-0.1 and E-10PC-0.1 M electrolytes. Additionally,the lower desolvation energy of K + solvation shell38 produces the very initial SEI film, diminishing PC decompositionand cointercalation, which is elaborately discussed in the later section.3.43 C – C/C–Hpeak (284.8 eV) and electrolyte solvent decomposition products, i.e.,C–O (∼285.7 eV), C=O (∼287.2 eV), O=C–O(∼288.7 eV), CO3 (∼289.5 eV) from EC [(CH2O)2CO ] and EMC [C2H5OCOOCH3 ] .32 The CH2 –CF2 (∼290.4 eV) and CF2 – CF2 (∼291.2 eV) peaks are from PVDF binder in the graphitecoating and its further fluorination, respectively,32 which are also identified at ∼687.3 and ∼688.3eV binding energy in the F 1s spectrum in 3/2 and K 2p1/2, respectively, which is confirmed by K 2speak at ∼378 eV in Figure S10. TheF 1s spectrum in xPFyOz .396 primary and KPF6 additive salts,respectively.Furthercharacterizations are employed to investigate the interphase chemistryof SEI formed on the graphite anode with modified electrolytes inorder to understand the mechanism behind enhanced cell performance. Table S3 shows the list of the ion clusters collected on the graphite anodecycled with PC-based ternary electrolytes. 7Li + and 19F – are the dominant peaks inpositive and negative ion spectra, as clearly evident by the correspondingSIMS maps in 2F + in 39K +, 41K + in Figure S11).The ion fragments associated with LiF are 6Li7Li + (32 amu), 7Li2F + (33 amu), 7Li3F + (40 amu), 7Li2F2+ (52 amu), 6Li7Li2F2+ (58 amu),and 7Li3F2+ (59 amu)observed in positive ion spectra in 6LiF2– (44 amu), 7LiF2– (45 amu), 7Li2F3– (71 amu) negative ion fragments in 45 In addition, K isotopes at 39 amu (39K + )and 41 amu (41K + ) are also present in positiveion mode (39K2F + (97 amu), 39K41KF + (99 amu), 39K3F2+ (155 amu), and 39K241KF2+ (157 amu) in positiveion and 39KF2– (77 amu), 41KF2– (79 amu), 39K2F3– (135 amu), and 39K41KF3– (137 amu)in negative ion spectra47 are not present, indicating theabsence of KF. Although two small peaks are 97 and 99 amu in positiveion spectra, the intensity ratio (99I/97I) does not match with the relative abundanceratio of potassium isotopes (39K/41K = 13.8).48 SIMS was also repeated at the same locationfor the number of cycles; however, the intensity ratio of 39K/41K does not match with the intensity ratio at 97 and99 amu, confirming these peaks are not 39K2F + and 39K41KF +, respectively. This indicates that KF isabsent as an SEI constituent. Therefore, the ion species at 97 and99 amu could be C5H5O2+ and C5H7O2+, respectively. This validates that the increase in concentrationpercentage of metal fluoride is due to higher LiF content in the optimizedelectrolyte; E-10PC-0.1 M.To gain better insight into the composition ofmetal fluoride,secondary ion mass spectroscopy (SIMS) was carried out, as shown in 50 inhibiting PC cointercalationand successive graphite exfoliation caused by this. The suppressionof PC decomposition is the dominant reason for improving the electrochemicalperformance, which is primarily associated with the following factors.6 additive enhances the fluoride(LiF) content in the SEI layer, investigated by XPS and SIMS spectrain 51 ensures the stabilityof the film in PC-based electrolyte upon cycling. This stable SEIprevents the cointercalation of PC solvent into the graphite layersand, therefore, suppresses the subsequent graphite exfoliation. Inaddition, the insulating nature of LiF52 blocks thepotential electron leakage from the defect sites,28 which inhibits any possible electrolyte decomposition aswell as Li dendrite formation/growth—facilitating faster Li + transportation through graphite anode.54 Furthermore, LiF, an inorganic SEI compound is highly probable tobe seen toward the graphite anode in the SEI layer (away from theelectrolyte). Hence, increased LiF content acts as a perfect SEI constituentin obstructing the PC intercalation in order to overcome the exfoliationof the graphite anode.0.1 M KPF+ (suchas Cs + ) exhibits a faster diffusion rate and lower desolvationenergy in the electrolyte compared to Li + solvation shell,38 facilitating the rapid formation of initial SEI layer at the electrode/electrolyteinterphase.24 Herein, K + solvationshell (due to lower Stokes radius compared to Li + ) producesthe very initial SEI layer with higher LiF content in PC-based electrolyte.As previously mentioned, the intrinsic properties of LiF restrictPC decomposition and its cointercalation into the graphite anode,hence suppressing the graphite exfoliation and Li dendrite formation,thereby improving the electrochemical performance of the cell.It has been previouslystudied that the solvation shellof cations having lower Stokes radius compared to Li LiF produces a compact and robustSEI film due to its lower solubility,A schematic illustration is shown in 46 as the main salt and KPF6 as an additive to acquire optimized properties of the solvents.The concentration of KPF6 electrolyte additive with volumepercentage of PC solvent is systematically investigated and analyzedalongside a commercial electrolyte. It is found that 0.1 M KPF6 suppresses not only the exfoliation of graphite layers butalso Li dendrite formation, maintaining the cyclability in PC-basedelectrolytes. The faster diffusion rate and lower desolvation energyof the K + solvation shell produce the very initial LiF-richSEI film, decreasing PC decomposition on the graphite surface, therebyminimizing the probability of graphite exfoliation. In addition, thestrong, insoluble SEI layer with higher LiF content maintains thestructural integrity of graphite anode in optimized PC-based electrolyte;i.e., E-10PC-0.1 M. Furthermore, the electron blocking capacity ofLiF-rich SEI layer eliminates the risk for Li dendrite formation,resulting in enhanced electrochemical performance.In summary, a ternary electrolyteis formulated with PC, EC, andEMC solvents along with LiPF


S100a16 knockdown alleviated ethanol-induced liver injury, steatosis and inflammation. Conversely, S100a16 transgenic mice showed aggravating phenomenon. Mechanistically, we identify mesencephalic astrocyte-derived neurotrophic factor (MANF) as a regulated entity downstream of S100a16 deletion. MANF inhibited ER-stress signal transduction induced by alcohol stimulation. Meanwhile, MANF silencing suppressed the inhibition effect of S100a16 knockout on ethanol-induced lipid droplets accumulation in primary hepatocytes. Our data suggested that S100a16 deletion protects mice against alcoholic liver lipid accumulation and inflammation dependent on upregulating MANF and inhibiting ER stress. This offers a potential therapeutic avenue for ALD treatment.Alcoholic liver disease (ALD) encompasses conditions ranging from simple steatosis to cirrhosis and even liver cancer. It has gained significant global attention in recent years. Despite this, effective pharmacological treatments for ALD remain elusive, and the core mechanisms underlying the disease are not yet fully comprehended. S100A16, a newly identified calcium-binding protein, is linked to lipid metabolism. Our research has discovered elevated levels of the S100A16 protein in both serum and liver tissue of ALD patients. A similar surge in hepatic S100A16 expression was noted in a Gao-binge alcohol feeding mouse model.  Alcohol-associated liver disease (ALD) has emerged as a significant global public health concern, exerting immense pressure on healthcare systems  S100a16 has shown potential in mitigating fatty liver injury. Our data further indicated that silencing of S100a16 can protect mice from liver fibrosis when exposed to stimuli such as CCl4, bile duct ligation surgery, and methionine choline deficiency diet feeding S100a16 knockdown against lipid accumulation and inflammation in HK-2 cells under high glucose conditions The S100 calcium-binding protein 16 (S100A16), a recently identified member of the EF-hand S100 family proteins, exhibits broad expression across diverse human tissues, notably in the liver and adipose tissue Endoplasmic reticulum stress (ER stress) is a cellular defense mechanism triggered in response to the accumulation of misfolded and unfolded proteins within the endoplasmic reticulum's lumen. This protective process initiates the unfolded protein responses (UPR). Three canonical UPR signaling pathways have been delineated: inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like ER kinase (PERK) Manf overexpression could directly promote browning of white adipocytes via the p38 MAPK pathway, thereby counteracting HFD-induced obesity Manf overexpression reduced lipid accumulation in liver, whereas Manf ablation exacerbated hepatic steatosis. Mechanistic insights revealed that MANF inhibition upregulated G0S2, a critical determinant that fostered lipid accumulation and suppressed lipolysis Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an emerging dopaminergic neurotrophic factor believed to have regulating potential for ER stress  S100a16 knockdown mitigated the severity of alcohol-induced fatty liver injury in mice. Mechanistically, the surge in S100A16 induced by alcohol was found to inhibit MANF, which in turn fostered lipid accumulation and inflammatory reactions. Collectively, our findings posited S100A16 as a potential novel regulator in the pathogenesis of ALD.In our current research, we observed a marked upregulation of S100A16 in ALD patients. Additionally,Pathological section of liver and serum from subjects with ALD and healthy controls were obtained from the first affiliated hospital of Nanjing Medical University . All ALD patients had history of alcohol consumption averaging at least 70 g/day, for at least 20 years. We excluded patients with drug-induced liver injury, autoimmune liver disease, hepatitis B or C, and hepatocellular carcinoma. Samples from relative healthy subjects were known to have no history of excessive drinking or chronic liver disease. The information of ALD patients and healthy individuals are shown in Table S100a16 knockout heterozygote (S100a16KO+/-) mice and S100a16 transgenic (S100a16TG) mice were generated as described previously S100a16 allele with floxed exon 2-3 was backcrossed into the C57BL/6 background. It was then bred with EIIa-Cre mice expressing the Cre recombinase to generate S100a16KO+/- in over 98% C57BL/6 background. During this process, we found that S100a16 homozygous (S100a16KO-/-) mutations were lethal. So, we used S100a16 knockout heterozygote (S100a16KO+/-) mice in this research.S100a16 mice cDNA was cloned into the pCAGGS plasmid, transgenic mice (S100a16TG mice) were generated by purified DNA fragments microinjection into the pronuclei of fertilized eggs of C57BL/6J.S100a16 knockout (S100a16LKO) mice were generated by crossing S100a16-floxed mice with Albumin-Cre transgenic mice (Alb-Cre) with both strains on the C57BL/6J background. S100a16-floxed (S100a16f/f) mice were mated with Alb-Cre mice to generate S100a16LKO heterozygote mice. Then, S100a16LKO mice and their littermate controls (S100a16f/f mice) were obtained by mating S100a16LKO heterozygote mice with S100a16f/f mice.Hepatocyte-specific All mice used in our experiments were identified by PCR analyses of the genomic DNA obtained from tail tips. All animal were housed in ventilated cages at the SPF facility in a temperature-controlled environment with a 12-hour light/dark cycle and had free access to water and diet. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (No. IACUC-2105059).Age-matched eight- to ten-week-old male mice were treated with either an isocaloric ethanol or pair-fed control diet following the protocol of NIAAA model Serum and liver triglyceride (TG), total cholesterol (T-CHO) and serum alanine transaminase (ALT), aspartate transaminase (AST) levels were determined using biochemical assay kits  according to the manufacturer's specifications.in situ perfusion digestion of livers using type IV collagenase , followed by separation using PERCOLL  and centrifugation. Isolated primary hepatocytes were maintained in DMEM medium supplemented with 10 % Fetal Bovine Serum  and 1 % penicillin/streptomycin . The culture medium was replaced daily.Mice primary hepatocytes were isolated by 2.AML12 hepatocytes, a non-transformed mouse hepatocyte cell line, was maintained in DMEM/F12 medium  supplemented with ITS liquid media supplement , 40 ng/mL dexamethasone  and 10 % FBS . Cells were treated with medium containing 50 mM ethanol for 24 h and changed intervals fresh medium at 12 h. All the cells were incubated at 37 °C in a humidified atmosphere containing 5% COS100a16 and Manf, the AML12 cell line was transfected with siRNA or a full-length plasmid using Lipofectamine® 2000 transfection reagent  according to the manufacturer's protocol. Knockdown and overexpression of Manf in S100a16LKO or S100a16TG mice primary hepatocytes were performed as above described. Negative scrambled control siRNA and empty vector plasmid (pcDNA3.1) were transfected into the control groups.For the knockdown and overexpression of TM NT T33517). Nitrocellulose membranes were blocked in TBST containing 5% skim milk for 2 h at room temperature. Then the membranes were incubated in primary antibodies overnight at 4 ℃, followed by incubation in the corresponding secondary antibodies. The relative expression levels of target proteins were quantified using ImageJ software (version 1.48). All primary antibodies used are listed in Total protein was extracted from liver tissues and cells using RIPA lysis buffer (Beyotime P0013) added with protease inhibitors (biosharp BL612A) and phosphatase inhibitors (biosharp BL615A). Protein samples were mixed with loading buffer (Beyotime P0298) and separated by 6-12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the proteins were transferred onto nitrocellulose membranes . The concentration of RNA was quantified by a Nanodrop2000 spectrophotometer . cDNA was synthesized using HiScript III RT SuperMix for qPCR  and detected using QuantStudio 7 system (Applied Biosystems). Relative mRNA expression levels were calculated using 2Fresh liver tissue was fixed in 4 % formaldehyde solution. Fixed liver tissues were embedded in paraffin, cut into 5 μm thick slices and stained with hematoxylin and eosin (H&E) following the standard protocol for analysis.Fresh liver tissue was embedded with OCT freeze embedding agent and frozen, and then cut into 5 μm thick slices. Oil-red was stained following the standard protocol for analysis. Slices were scanned by an automatic digital slide scanner.Primary hepatocytes and AML 12 cells were first fixed with 4% paraformaldehyde followed by incubation with BODIPY® 493/503 (Invitrogen 2295013) for lipid staining. Cell nuclei were counterstained with DAPI . The lipid droplets were visualized under a THUNDER Imaging Systems .Liver tissue slices were permeabilized with 0.5 % Triton X-100 for 30 min (after deparaffinization) and blocked with 5 % goat serum for 1 h at RT. Then the slices were incubated with anti-S100A16 or anti-MANF primary antibodies overnight at 4°C, followed by an appropriate fluorescently tagged secondary antibody in the dark at RT for 1.5 h. Primary and secondary antibodies were diluted in 3 % BSA. DAPI was used to stain nuclei. Fluorescence microscopy was performed using a THUNDER Imaging Systems .Commercially available human ELISA kits  were used to detect S100A16 levels in human serum  according to the instructions provided by the manufacturer.S100a16KO+/- mice. Total RNA was extracted, and mRNA was purified. mRNA was fragmented and reverse transcribed to generate cDNA. Sequencing analysis was performed as our previous work p value < 0.05 and fold change ≥ 2 were considered differentially expressed using DESeq2 software between S100a16 silencing group and WT group. Gene functional annotations were assessed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. The raw sequence data have been submitted to the NCBI Gene Expression Omnibus (GEO) database (accession no. GSE243683).Primary hepatocytes (n=3/group) were isolated from alcohol-fed WT and p < 0.05 was considered statistically significant.All data are expressed as the mean ± SEM. Two-tailed student's t-test was used to calculate the statistical significance between two groups and one-way analysis of variance (one way ANOVA) for three or more groups. In all cases, Fig. A, S1A. Concurrently, serum S100A16 levels were found to be elevated in individuals diagnosed with ALD Fig. B. Subsequently, we employed a short-term chronic plus one binge ethanol feeding model (NIAAA model). Analyses using western blot, RT-qPCR, and immunofluorescence staining collectively highlighted the heightened expression of S100A16 in the livers of alcohol-fed WT mice Fig. C-E. In parallel, in vitro experiments illustrated that ethanol exposure upregulated S100A16 expression in primary hepatocytes as well as AML12 cells Fig. F-I. Overall, these results suggested that alcohol consumption significantly increased hepatic S100A16 expression.To investigate the potential involvement of S100A16 in ALD progression, we first evaluated S100A16 expression levels in patients with ALD and healthy controls. Both immunofluorescence and immunohistochemical assays indicated S100A16 was significantly increased in the ALD liver tissue compared with healthy individuals S100a16 knockout mice. Homozygous deletion of S100a16 in mice causes mortality, therefore we used S100a16 heterozygous knockout mice (S100a16KO+/- mice). Genotyping of S100a16KO+/- and wild type (WT) mice were performed before the experiment A. Deletion of S100A16 in the liver of S100a16KO+/- mice was confirmed at the protein level B. Our data showed that the livers became bigger in the WT group after ethanol exposure, but this condition was alleviated in alcohol-fed S100a16+/-KO mice Fig. A. Western blot and RT-qPCR assays verified the augmented S100A16 expression in the livers of alcohol-fed WT mice Fig. B-C. While body weights remained consistent between alcohol-fed S100a16KO+/- and WT mice C, but liver/body weight ratios were mitigated in S100a16+/-KO group Fig. D. As shown in Fig. , knockout of S100a16 alleviated the upregulation of serum ALT, AST levels during alcohol consumption. Serum TG, TC contents and liver TG, TC contents all showed a decline in the S100a16KO+/- group Fig. F. Liver histological studies, encompassing H&E and Oil Red O staining, pointed to a mitigated liver injury in the S100a16KO+/- cohort Fig. G. Additionally, there was a marked suppression in the expression of crucial lipogenic and inflammatory genes in the livers of these mice upon genetic S100a16 inhibition Fig. H-I. Also noteworthy, lipid droplets in ethanol-induced primary hepatocytes isolated from S100a16KO+/- mice showed a significant reduction when compared with that from WT mice Fig. J. As shown in , we employed siRNA to diminish S100A16 expression (S100A16 KD) in AML12 cells and the knockdown efficiency was confirmed using western blot. Analogous to our earlier findings, S100a16 knockdown led to diminished lipid droplets in AML12 cells Fig. K. In summary, inhibiting S100A16 substantially alleviated the symptoms of alcohol-induced fatty liver injury.To elucidate the relationship between S100A16 and alcoholic fatty liver disease, we generated S100a16 transgenic (S100a16TG) mice systemically overexpressing S100a16 were engineered under control of the pCAGGS vector promoter A. Expression of S100A16 in the liver of S100a16TG mice was confirmed at the protein level B. We then exposed age-matched WT and S100a16TG mice to the NIAAA model, observing the subsequent changes. Unlike the results from S100a16KO+/- mice, post-ethanol exposure, the livers of S100a16TG mice had larger livers than WT mice after ethanol exposure Fig. A. Moreover, liver-to-body weight ratios surged in the S100a16TG group Fig. D. Results showed that S100A16 overexpression in liver was increased in the alcohol-fed mice Fig. B-C. Contrasting with the effects of S100a16 suppression, overexpression of S100a16 exacerbated the progression of alcohol-induced fatty liver development compared with WT mice. This exacerbation was evident both biochemically  and histologically (using H&E and Oil Red O staining) Fig. E-G. The mRNA levels of molecules that promote fat accumulation and inflammation were also augmented in alcohol-exposed S100a16TG mice Fig. H-I. Notably, ethanol-induced primary hepatocytes derived from S100a16TG mice exhibited a pronounced increase in lipid droplets compared with primary hepatocytes from WT mice Fig. J. As shown in , S100A16 overexpression efficiency in AML 12 cells was confirmed by western blot analysis. Similarly, overexpression of S100A16 in AML12 cells also amplified ethanol-induced lipid accumulation Fig. K. Collectively, these findings compellingly argued that overexpressing S100A16 intensified the manifestations of alcohol-induced hepatic steatosis and injury.To further verify the relationship between S100A16 and alcoholic fatty liver injury diseases, S100a16KO+/- mice subjected to NIAAA model and genome-wide analysis of gene expression was performed by transcriptome sequencing. Those detectable genes with a fold change ≥ 2 and p value < 0.05 were defined as differentially expressed genes, when compared with equivalents measured from WT cells. Consequently, S100a16KO+/- cells gave rise to 550 differentially expressed genes . The PCA plot is presented in . Utilizing GO and KEGG enrichment analyses, we discerned the distribution of these differentially expressed genes, uncovering that the absence of S100a16 significantly affected intracellular processes, including the endoplasmic reticulum stress (ER stress) pathway Fig. B. We then constructed a protein-protein interaction network B and identified hub genes using STRING (https://stringdb.org/) and Cytoscape (version 3.8.0). Notably, among these hub genes Fig. C, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been implicated as pivotal in alcohol-induced liver diseases Fig.  we verified the RNA sequencing result using RT-qPCR. In mice liver tissue, we also found that gene and protein expression of MANF were increased in WT mice after alcohol feeding and these alterations were further increased in S100a16KO+/- mice Fig. E-G. Conversely, a decline in MANF gene and protein expression was observed in alcohol-fed S100a16TG mice Fig. H-J. From these findings, we postulated that S100a16 suppression ameliorated alcoholic fatty liver injury by augmenting MANF expression.Subsequently, we examined the underlying mechanism of S100A16 in regulating alcoholic fatty liver injury. Primary hepatocytes were isolated from alcohol-fed WT or S100a16f/f mice were crossed with Albumin-Cre mice to generate hepatocyte-specific S100A16 knockout mice (genotype: S100a16LKO) and control mice (genotype: S100a16f/f) A-B. Deletion of S100A16 in the liver of S100a16LKO mice was confirmed at the protein level C. mRNA and protein levels of MANF were increased in the livers from alcohol-fed S100a16LKO mice compare with S100a16f/f mice D-E. Similar with S100a16KO+/- mice, liver/body weight ratios were alleviated in S100a16LKO group F. As depicted in , the specific knockout of S100A16 in hepatocytes attenuated alcohol-induced fatty liver development, as demonstrated by both biochemical assay  and histological examination (H&E staining). We also observed that hepatocyte-specific genetic suppression of S100a16 significantly curtailed the expression of key lipogenic and inflammatory genes in mice livers J-K.To more accurately confirm the role of S100A16 in alcoholic fatty liver disease, S100a16KO+/-mice livers, there was a pronounced reduction in the expression of ER stress markers such as GRP78, ATF4, ATF6, and in the phosphorylation levels of IRE1α and EIF2α compared with their WT counterparts Fig. A. Conversely, these trends were inverted in the S100a16TG mice Fig. B. In summation, S100a16 ablation augmented MANF expression, thereby curtailing ER stress, which in turn mitigates the progression of alcoholic fatty liver development.MANF, situated within the ER lumen, serves as a crucial shield against ER stress-induced cellular damage S100a16LKO mice. RT-qPCR analyses authenticated the deletion of both S100A16 and MANF in primary hepatocytes Fig. A. Observations from BODIPY staining indicated that Manf siRNA suppressed the inhibiting effects of S100a16 deletion on fat accumulation Fig. B. Double knockdown of Manf and S100a16 in AML12 cells also confirmed these results Fig. C-D. Elevated TG levels further substantiated that double knockdown of Manf and S100a16 in primary hepatocytes/AML12 cells inhibited the lipid-reducing impact of S100a16 deletion A-B. Conversely, we induced overexpression of Manf in primary hepatocytes from S100a16TG mice, RT-qPCR analyses confirmed elevated levels of both S100a16 and ManfFig. E. Results showed that MANF overexpression significantly counteracted the lipid-enhancing effects of S100a16 overexpression Fig. F. This observation was also corroborated in AML 12 cells Fig. G-H. TG levels showed the same results C-D. In essence, these outcomes suggested that the protective role of S100a16 deletion protected hepatocytes against alcohol-induced lipid accumulation dependent on promoting MANF expression.To ascertain whether S100A16 mediated ethanol-induced lipid accumulation dependent on MANF, we silenced MANF in primary hepatocytes isolated from  Fig. .Alcohol abuse stands as a prime cause of liver-related mortalities globally. Despite its grave implications, there remains an absence of efficacious treatments beyond lifestyle adjustments S100 proteins traditionally influence myriad cellular functions, from differentiation and cycle progression to motility S100a16KO+/- mice compared with cells from WT. We further confirmed that MANF expression in liver was markedly promoted in alcohol-fed S100a16KO+/- mice. Reverse results were observed in alcohol-fed S100a16TG mice livers. Therefore, we speculated that S100A16 downregulated MANF expression and promoted alcohol-induced liver lipid accumulation and inflammation responses. In addition, MANF silencing abrogated the inhibitory effects of S100a16 knockout on lipid droplets formation in primary hepatocytes isolated from S100a16LKO mice in ethanol-containing medium. In contrast, MANF overexpression abrogated the protective effects of S100a16 transgene on lipid droplet formation. Similar results were confirmed in AML12 cells cultured medium containing ethanol. These results provided evidences that S100a16 deletion alleviates alcoholic fatty liver injury dependent on upregulating MANF expression. Besides, we also evaluated how S100A16 regulate MANF expression. We found S100A16 inhibited MANF expression using RNA sequencing, so we speculated that S100A16 transcriptionally regulated MANF. Indeed, S100A16 co-transfection decreased the luciferase activity of MANF promoter E. A cycloheximide (CHX) chase assay was performed in S100A16 plasmid-transfected AML 12 cells to investigate the effect of S100A16 on the degradation rate of MANF protein. When protein synthesis was inhibited with CHX to exclude the potential effects, MANF protein was degraded in a time-dependent manner and S100A16 overexpression resulted in faster degradation, indicating that S100A16 promoted MANF degradation F. Based on these results, we assumed that S100A16 inhibited MANF expression both by inhibiting its mRNA transcription and accelerating protein degradation process.Another major finding of this study is that we identified MANF, an endoplasmic reticulum-resident protein, as the downstream of S100A16 in alcoholic liver lipid accumulation. MANF is an emerging therapeutic target for metabolic diseases, including diabetes ER stress participated in the regulatory process of many diseases, including metabolic disorders It has been reported that males are more likely than females to consume alcohol and males are diagnosed with alcohol use disorders more often than females In summation, our insights underscore S100A16 as a novel ALD regulatory factor, affecting MANF expression and exacerbating ER stress. This points to S100A16's potential as a promising therapeutic bullseye for ALD.Supplementary materials and methods, figures and tables.Click here for additional data file.


Background: The purpose of this study was to clarify the effect of C-X-C chemokine receptor type 7 (CXCR7) on proliferation, migration, and angiogenesis by changing the expression levels of CXCR7 in colon cancer cells. Contrast-enhanced ultrasound technology was used to quantify tumor perfusion parameters in vivo for the detection of angiogenesis after the change of CXCR7 expression in colon cancer xenografts.Methods: To detect the expression of CXCR7 in colon cancer cells after overexpression or silencing of CXCR7. In addition, proliferation, migration, and angiogenesis were determined. The region of interest of the tumor was selected, and a time-intensity curve was drawn. Immunohistochemical staining was performed on tumor tissue sections, and the average microvessel density value was calculated.Results: Overexpression or silencing of CXCR7 altered the proliferation, migration, and luminal formation of Caco-2 and SW480 cells. In xenografts produced using CXCR7-overexpressing or -silent Caco-2 and SW480, respectively, the peak intensity and area under the curve were significantly different. The expression of CXCR7, VEGF, Ki67, and CD34 was decreased in CXCR7-silent cells, but increased in CXCR7-overexpressing cells. CXCR7 apparently affected angiogenesis through the extracellular signal regulated kinase pathway.Conclusions: The regulation of CXCR7 expression may affect the proliferation, migration, and luminal formation of Caco-2 and SW480 cells, indicating that CXCR7 may play an important role in colon cancer. Examination through contrast-enhanced ultrasound also demonstrated that the expression of CXCR7 is closely related to angiogenesis. C-X-C chemokine receptor type 4 (CXCR4) was previously thought to be the only receptor for C-X-C motif chemokine ligand 12 (CXCL12). However, since the discovery of CXCR7in vivo. WangContrast-enhanced ultrasound (CEUS) is a rapid and easy assessment method. It can be repeatedly performed in patients, and has high spatial and temporal resolution, providing an excellent opportunity for the visualization of microcirculation. It can display the shape and distribution of tumor blood vessels in real time, and be used to evaluate tumor angiogenesis. The use of CEUS to quantify tumor perfusion for the early detection of tumor angiogenesis has been previously described in the literatureCaco-2 and SW480 colon cancer cell lines and human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences . Caco-2 and SW480 cell lines were maintained in DMEM  with 10% fetal bovine serum . The HUVECs were maintained in PRMI 1640 medium (HyClone) containing 10% fetal bovine serum . All cells were cultured at 37°C and 5% CO2 in a humidified incubator.CXCR7OE) and pCMV6 Entry vector (Caco-2CXCR7NC) , respectively. For the CXCR7-silent colon cancer group and its NC group, SW480 cells were transfected with short hairpin RNA (shRNA) (SW480shRNACXCR7) or unrelated sequences (SW480shRNANC) , respectively. SW480 and Caco-2 cells without any treatment (Control). All cells were transfected with Lipofectamine 3000  and screened for stably transfected cells with G418 . The cells were subsequently cultured in an incubator with 5% CO2 at 37°C. The shRNA and control shRNA sequences are as follows: CXCR7: shRNA, 5′-GATCCCCGAGCTCACGTGCAAAGTCATTCAAGAGATGACTTTGCACGTGAGCTCTTTTT‐3′ (sense) and 5′‐AGCTAAAAAGAGCTCACGTGCAAAGTCATCTCTTGAATGACTTTGCACGTGAGCTCGGG‐3′ (antisense). NC: 5′‐GATCCCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTT‐3′ (sense) and 5′‐AGCTAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAGGG‐3′ (antisense).For the CXCR7-overexpressing colon cancer group and its negative control (NC) group, Caco-2 cells were transfected with the pCMV6 Entry-CXCR7 plasmid (Caco-2Used TRIzol reagent (Invitrogen) to extract the total RNA of the sample, and reverse transcribed in accordance with the instructions provided in the reverse transcription kit . The primers used in this experiment were as follows: CXCR7, 5'-TCTGCATCTCTTCGACTACTCA-3' (forward) and 5'-GTAGAGCAGGACGCTTTTGTT-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-GAAGGTGAAGGTCGGAGT-3' (forward) and 5'-GAAGATGGTGATGGGATTTC- 3' (reverse). The expression of mRNA was calculated based on the 2-ΔΔCt method.Total protein lysates were prepared using the radioimmunoprecipitation assay buffer  for 15 min. Used the bicinchoninic acid protein concentration assay kit (Beyotime) to calculate the protein concentration of the sample. The dilution ratio of the anti-CXCR7 anti-GAPDH antibodies  was 1:1,000 and 1:2,000, respectively. The cells were incubated with the primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Abcam) . Visualization was achieved through chemiluminescence.Each group of Caco-2 or SW480 cells was seeded in 96-well plates with 3,000 cells per well. After culturing for 24, 48, and 72 h, 100 μL of medium containing 10% CCK-8  was added to each well at 37°C for 2 h, and the absorbance was measured at 450 nm . Prepared an appropriate amount of 50 μM EdU medium, and added 200 μL of medium to each well for 2 hours. The wells were subjected to Apollo staining in accordance with the instructions provided by the manufacturer, and photographed using a fluorescence microscope .5cells per well. Subsequently, 500 μL of medium containing 20% FBS was added to the lower chamber, and the cells were incubated at 37°C for 6 h. A microscope (Nikon) was used to count the number of cells under the membrane surface in five different fields of view at ×200 magnification after fixation and staining.The transfected cells were seeded into the upper chamber with 4×105cells/well) were seeded in the upper chamber, while human umbilical vein endothelial cells  were seeded in the lower chamber coated with Matrigel™. After incubation at 37°C for 6 hours, the tube formation was counted and photographed with a microscope (Nikon).The Matrigel™ tube formation assay was used to measure the ability of endothelial cells to arrange into tubular structures. Matrigel™ (200 μL)  was spread in the lower chamber and incubated at 37°C for 30 min. Subsequently, the transfected cells  in FBS. The mice were maintained for 4-6 weeks. CEUS examination was performed following tumor growth to approximately 1 cm3. After CEUS examinations, the mice were euthanized by CO2 inhalation. Animal studies were in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council and approved by the China Medical University Animal Ethics Committee (IACUC Issue No.16071). All procedures accepted the supervision and inspection by the committee and laboratory animal department.Female nude mice, aged 4-6 weeks  were purchased and raised in a specific-pathogen-free environment. Each nude mouse  was subcutaneously injected into the right lower extremity with 0.2 mL of a cell suspension containing 1×10Conventional two-dimensional ultrasound and CEUS were performed using the clinical diagnostic ultrasound system Aplio 500  with a 10-MHz superficial probe. A specific calibration file  provided by the vendor was used as the analysis software. Ultrasound images were converted into linearized data for time-intensity curve (TIC) analysis. Firstly, the tumor size and morphology were carefully observed via two-dimensional ultrasound, and the color flow distribution was determined using color Doppler flow imaging. The acoustic contrast agent SonoVue  was used. The region of interest of the tumor was selected, and 100 µL of ultrasound contrast agent was injected into the tail vein of each mouse. Subsequently, the ultrasound contrast image and the original data were acquired. All ultrasound images were saved in a computer, and a TIC was constructed for the region of interest of the specified image using the system's own image analysis software. The following perfusion parameters were obtained: PI, time to peak (TTP), mean transit time (MTT), AUC, and slope.After acquiring the CEUS images, each mouse was euthanized and the tumor tissue was removed. Tumor tissue sections corresponding to the ultrasound imaging plane were prepared for immunohistochemical staining. The sections were fixed in 10% formalin, embedded in paraffin, cut into slices (thickness: 4 µm), dewaxed with xylene, and immersed in 100%, 95%, 85%, and 75% alcohol for 5 min. The sections were quenched with 3% hydrogen peroxide for 10 min, and incubated for 10 min in protein blocking solution . They were subsequently incubated with primary antibody overnight at 4°C. The primary antibodies and dilution concentrations were as follows: CXCR7 antibody (1:200), CD34 antibody , ki67 antibody (1:100), VEGF antibody (1:100), extracellular signal regulated kinase (ERK) antibody (1:100), and p-ERK antibody (1:500) (Abcam). The blots were probed with antibodies specific for ERK phosphorylation at Thr202 and Tyr204. After addition of the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG [Abcam]) and incubation for 2 h, diaminobenzidine was added dropwise for color development. Brown staining indicated positivity for the primary antibody. Immunohistochemical results were scored to determine the intensity of positive staining and the proportion of tumor cells.MVD measurements (the gold standard for assessing tumor angiogenesis) were determined according to the quantitative method described by WeidnerData analysis and processing were performed using the SPSS 19.0  statistical software. One-way analysis of variance was used for comparison between groups. Spearman's rank correlation analysis of CEUS perfusion parameters and MVD was performed; p<0.05 denoted statistically significant difference.The expression of CXCR7 in colon cancer cell lines  was evaluated by qRT-PCR and western blotting (Figure The expression of CXCR7 in CXCR7-silent SW480 cells and CXCR7-overexpressing Caco-2 cells was detected by qRT-PCR and western blotting. The results showed that the protein expression and mRNA levels of CXCR7 in CXCR7-silent SW480 cells were markedly reduced compared with those of the NC group Figure B. In conWe investigated the effect of CXCR7 overexpression or silencing on the proliferation of colon cancer cells. For this purpose, EdU and CCK-8 were used to detect the proliferative activity of CXCR7-overexpressing or -silent colon cancer cells. The number of EdU-positive cells among CXCR7-silent SW480 cells was markedly reduced. In contrast, the number of EdU-positive cells among CXCR7-overexpressing Caco-2 cells was markedly increased Figures D and 1E.The results of the Transwell migration experiment showed that the migratory ability of CXCR7-silent SW480 cells was lower than in the NC group Figure A. On theCXCR7-silent SW480 cells and CXCR7-overexpressing Caco-2 cells were co-cultured with HUVEC. The lumen-forming ability of HUVEC on Matrigel was evaluated by measuring the number of junctions. After co-culture with CXCR7-silent SW480 cells, the angiogenic ability of HUVEC was markedly reduced (Figure The examination using two-dimensional ultrasound showed that the transplanted tumors were generally elliptical or irregular, and their boundaries were clear. The interior had a medium or low echo, and the distribution was not uniform Figure A. The coImmunohistochemical staining for CXCR7, VEGF, Ki67, and CD34 was reduced in the CXCR7-silent SW480 group. We further investigated the role of the ERK pathway in angiogenesis. The results showed that antibody staining of p-ERK in the CXCR7-silent group was significantly reduced with lower staining scores (Figure In contrast, immunohistochemical staining for CXCR7, VEGF, Ki67, and CD34 was increased in the CXCR7-overexpressing Caco-2 cells group. Antibody staining of p-ERK after CXCR7 overexpression was significantly increased with higher staining scores Figure A and 5B.The expression of VEGF, ERK, and p-ERK in CXCR7-silent SW480 cells and CXCR7-overexpressing Caco-2 cells was detected by western blotting. The results showed that the protein expression of VEGF and p-ERK in CXCR7-silenced SW480 cells were markedly reduced A. In conNext, we investigated the relationships between the perfusion parameters and MVD in tissues with silenced and overexpressed CXCR7 Table . In SW48in vitro, and promotes tumor growth in vivoin vitro, thereby promoting tumor growth.A growing body of evidence indicates that CXCR7 may be involved in tumor development. CXCR7 was highly expressed in various tumor cellsCEUS is a major development in ultrasonographyPrevious studies have shown that the ERK pathway is important for tumor survival, proliferation, and angiogenesisSupplementary figure.Click here for additional data file.


Correction: J Ovarian Res 11, 3 (2018)10.1186/s13048-017-0377-5Figure 3A has been corrected.Figure 7d has been corrected.Figure S5a legend was revised to include the reference to a previous paper as shown below.The original article  containsFigure S5a. Z stack of OCT-4 expressing germ cell clusters in FSH treated OSE cell culture [ culture .The correct Figs. The authors sincerely apologize for the errors. The errors do not affect the conclusion of the article.


CDKN2A and CDKN2B in astrocytomas have been an evolving area of study for decades. Most recently, there has been considerable interest in the effect of CDKN2A and/or CDKN2B (CDKN2A/B) homozygous deletions (HD) on the prognosis of isocitrate dehydrogenase (IDH)-mutant astrocytomas. This is highlighted by the adoption of CDKN2A/B HD as an essential criterion for astrocytoma and IDH-mutant central nervous system (CNS) WHO grade 4 in the fifth edition of the World Health Organisation (WHO) Classification of Central Nervous System Tumours (2021). The CDKN2A and CDKN2B genes are located on the short arm of chromosome 9. CDKN2A encodes for two proteins, p14 and p16, and CDKN2B encodes for p15. These proteins regulate cell growth and angiogenesis. Interpreting the impact of CDKN2A/B alterations on astrocytoma prognosis is complicated by recent changes in tumour classification and a lack of uniform standards for testing CDKN2A/B. While the prognostic impact of CDKN2A/B HD is established, the role of different CDKN2A/B alterations—heterozygous deletions (HeD), point mutations, and promoter methylation—is less clear. Consequently, how these alternations should be incorporated into patient management remains controversial. To this end, we reviewed the literature on different CDKN2A/B alterations in IDH-mutant astrocytomas and their impact on diagnosis and management. We also provided a historical review of the changing impact of CDKN2A/B alterations as glioma classification has evolved over time. Through this historical context, we demonstrate that CDKN2A/B HD is an important negative prognostic marker in IDH-mutant astrocytomas; however, the historical data is challenging to interpret given changes in tumour classification over time, variation in the quality of evidence, and variations in the techniques used to identify CDKN2A/B deletions. Therefore, future prospective studies using uniform classification and detection techniques are required to improve the clinical interpretation of this molecular marker.Genomic alterations of  Prior to 2016, many studies classified tumours based only on morphology. Consequently, tumours previously classified as astrocytomas on morphological grounds are likely to include tumours that are currently classified as astrocytoma, IDH-mutant, oligodendroglioma IDH-mutant, 1p/19q codeleted or glioblastoma, and IDH-wildtype ,16,17. ICurrently, there are no consensus treatment protocols for IDH-mutant CDKN2A/B HD astrocytomas. A significant proportion (6–20%) of grade 4 IDH-mutant astrocytomas with CDKN2A/B HD were previously classified as either grade 2 or grade 3 tumours ,11,12,13To guide understanding and management of this newly defined group, we performed a literature review on CDKN2A/B HD in astrocytomas see . We presCDKN2A and CDKN2B are adjacent to each other on chromosome 9p21, with CDKN2B being 25 kilobases (kb) centromeric to CDKN2A  and is located approximately 100 kb telomeric to CDKN2A. MTAP is required for adenosine monophosphate and methionine salvage and has a tumour suppressive effect in multiple cancers . Th. ThCDKN2CDKN2A/B HD. These include single-nucleotide polymorphism (SNP) microarrays [A variety of methods can be used to evaluate roarrays ,53, nextroarrays ,29, DNA-roarrays ,18,19,20roarrays ,28. It sCDKN2A and CDKN2B in the assessment of HD [Although SNP arrays, NGS, and methylation arrays possess greater resolution for individual gene-level detection, many studies combine nt of HD ,29,31,53nt of HD  and wholnt of HD ,18,19,20CDKN2A, CDKN2B, and MTAP genes [Fluorescence in situ hybridization (FISH) can be used to detect deletions and has been validated against methods utilising polymerase chain reaction (PCR) . ThreshoAP genes . TherefoCDKN2A HD in gliomas with mixed results. Given the close proximity of the CDKN2A and MTAP genes (see MTAP immunoreactivity has been suggested as a surrogate for CDKN2A HD [CDKN2A deletion [IDH-mutant astrocytomas [Immunohistochemistry (IHC) has been used to identify enes see , loss ofDKN2A HD  and has DKN2A HD ,58. In gdeletion . Howeverocytomas . IDH-mutant astrocytomas [CDKN2A HD in adult and paediatric morphologic glioblastomas [CDKN2A discordance with the IHC method [CDKN2A HD have been reported as 78–94% and 70–82%, respectively [CDKN2A/B deletions may be related to the additional loss of nearby genes, as noted above. Therefore, some tumours that are CDKN2A/B intact but have suppressed CDKN2A/B expression will have no immunoreactivity on p16 IHC but retain other genes that may confer a less aggressive phenotype. This means loss of CDKN2A/B in molecular studies may be more informative for prognosis than loss of p16 staining on IHC. However, Satomi et al. did show that loss of p16 immunoreactivity correlated with clinical outcome in ocytomas . While tlastomas , other sC method . Sensitiectively . FurtherCDKN2A/B deletions need to be interpreted in the context of the WHO classification used at that time  and CDKN2A HeD in 25% [CDKN2A HD, 3 with a heterozygous deletion, and one with a point mutation [Initial studies by Schmidt et al.  and GianN2B. see . Giani aD in 25% . Moultonmutation . Initial studies assessing the clinical impact of CDKN2A/B HD on prognosis in astrocytomas yielded conflicting results see . This waCDKN2A/B and biologic markers of tumour aggressiveness (tumour grade and Ki-67 index). Sonoda et al. suggested CDKN2A/B deletions may have a role in gliomagenesis and therefore more aggressive tumour biology. Using single-strand conformation polymorphism (SSCP) and quantitative polymerase chain reaction (qPCR), they showed an increased incidence of CDKN2A/B HD in high-grade gliomas  compared to low-grade gliomas  [CDKN2A/B HD in 50 astrocytomas and found a positive correlation between the Ki-67 index and CDKN2A HD (5/20 grade 3 astrocytomas and 6/13 glioblastomas had CDKN2A HD). CDKN2A HD was not identified in 17 grade 2 astrocytomas [CDKN2A HD in higher vs. lower grade tumours . They also showed a similar incidence of CDKN2A HD in the recurrent tumours (78%) [CDKN2A HD was associated with higher-grade tumours. Initial studies described the relationship between  = 1/10) . Buildinocytomas . Using Srs (78%) . These sCDKN2A HD was a negative prognostic factor in a heterogeneous group of gliomas that included anaplastic astrocytomas, oligoastrocytomas, and oligodendrogliomas. Although 1p/19q status was assessed, the authors did not identify which cases had CDKN2A HD and 1p/19q co-deletion [CKDN2A HD and clinical outcome [CDKN2A deletion in patients older than 50 years. Although IDH status was not reported in the study, this population was likely enriched for IDH-wildtype tumours, and it was later shown that CDKN2A deletions lack prognostic impact in these tumours [CDKN2A loss [CDKN2A deletion [In 2006, Dehais et al. reported that deletion . However outcome ,67. This tumours . The othdeletion .CDKN2A HD as a negative prognostic factor by assessing 135 gliomas for PTEN and CDKN2A copy number status. They reported an increasing frequency of CDKN2A deletions with grade . They observed that CDKN2A deletions were negative prognostic indicators of survival in all 135 gliomas, but this was not seen when stratifying for grade 3 or 4 separately [James et al. further supported parately . This laCDKN2A deletions increased, there was further focus on glioma subtypes, including oligodendrogliomas. Cairncross et al. explored the role of CDKN2A deletions in oligodendrogliomas using 1p/19q co-deletion instead of morphologic criteria alone. They analysed 39 morphologic oligodendrogliomas . Losses involving both chromosomes 1p and 19q were strongly associated with longer overall survival, whereas CDKN2A deletions were independent poor prognostic factors. The authors were able to analyse 34 of the 39 samples and noted that 22 patients had 1p/19q co-deletion. It is therefore assumed that the remaining 12 cases would most likely be astrocytomas by current classification [CKDN2A-deleted gliomas was less than 2 years and occurred preferentially in gliomas without loss of 1p or 19q [As interest in fication ,17 The oIDH-mutant astrocytomas. After reviewing the literature on multiple potential prognostic biomarkers, including CDKN2A/B HD, other Rb pathway genes, PIK3R1 and PIK3CA mutations, PDGFRA and MYCN amplification, reduced global DNA methylation, genomic instability (high copy number variants or somatic mutations), and mitotic activity and proliferation indices, they concluded that while “significant mitotic activity” should remain as a criterion for distinguishing grade 3 from grade 2 IDH-mutant astrocytomas, if CDKN2A/B HD, necrosis, or microvascular proliferation was present, a grade 4 designation was appropriate [In 2020, the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy (cIMPACT-NOW), upgrade 5, published recommendations for grading criteria and terminologies in ropriate .CDKN2A/B HD in grading IDH-mutant astrocytomas, several conflicting reports have been published. These recommendations were incorporated into the fifth edition of the WHO Classification of Tumours of the Central Nervous System 2021 . While tCDKN2A deletions as a prognostic marker specifically in IDH-mutant grade 2 and 3 gliomas. The authors analysed 270 gliomas and identified CDKN2A deletions via FISH in 57/108 grade 2 astrocytomas, 31/61 grade 3 astrocytomas, 23/96 oligodendrogliomas, and 19/49 oligoastrocytomas, inclusive of both homozygous and heterozygous CDKN2A deletions. The authors assessed tumours for 1p/19q deletion if they were not morphologic astrocytomas and assessed all tumours for IDH1/2 mutations by genome sequencing. They reported worse overall survival in grade 2 and 3 gliomas after adjusting for age, sex, and IDH mutation . This significance was maintained in the astrocytoma subgroup  but not for oligodendrogliomas or oligoastrocytomas . Again, a portion of these morphologic oligodendrogliomas in this cohort would no longer be classified as such without the corresponding molecularly confirmed 1p19q co-deletion. Interestingly, the presence of deletions in the IDH-mutant/ATRX expression loss astrocytoma group, without TP53 mutation, was non-prognostic (p = 0.2) [ATRX loss and TP53 mutations are strongly associated with IDH-mutant astrocytomas, it is unclear what this ATRX/TP53 discordance represents in IDH-mutant gliomas. Interestingly, given the FISH probe used covers a broad genomic region at 9p21, CDKN2B status can be said to be assessed by proxy. This study is therefore one of the few that demonstrates the prognostic role of CDKN2B [In 2015, Reis et al. identified p = 0.2) . Furtherf CDKN2B . IDH-mutant status [IDH-mutant astrocytomas, clinical outcomes for grade 2 and 3 IDH-mutant diffuse astrocytomas remained heterogenous [In 2016, the WHO Classification of Tumours of the Central Nervous System officially recognised that the diagnosis of oligodendrogliomas now required 1p/19q co-deletion in addition to rogenous ,63,68,69CDKN2A deletions, CDK4 amplification, and chromosome 14q loss were prognostic markers. Using these, they stratified IDH-mutant glioblastomas into 3 prognostically relevant molecular subgroups: M1 with chromosome 14q loss and either CDK4 amplification or CDKN2A deletion; M2 with either CDK4 amplification, CDKN2A deletion, or chromosome 14q loss; and M3 with no 14q loss, CDK amplification, or CDKN2A deletion. The median overall survivals for M1, 2, and 3 were 23.3, 63.0, and 94.5 months, respectively (p < 0.05) [CDKN2A HD can interact with other genomic alterations [To resolve this, Cimino et al. used The Cancer Genome Atlas (TCGA) sequencing datasets for glioblastomas, astrocytomas, and oligodendrogliomas to identify prognostic molecular markers. They found that  < 0.05) . This waerations .IDH-mutant astrocytoma samples. The findings were then validated using three independent cohorts of 108, 154, and 224 IDH-mutant astrocytomas. CDKN2A/B status was evaluated via the analysis of DNA-based methylation data. In the initial discovery cohort, they found 38 CDKN2A/B HD tumours. On univariate analysis, there was a significant negative correlation with OS and CDKN2A/B HD (p = 0.0001). They proposed three prognostic models for OS based on multivariate analysis of a discovery set and confirmation in three validation sets. In all models, tumours were classified as grade 4 if CDKN2A/B HD was present [CDKN2B HD in addition to CDKN2A HD and is one of only three studies in the Post-Molecular Characterisation Era to include both CDKN2A and CDKN2B deletions  and 483 anaplastic oligodendrogliomas (1p/19q co-deleted) using a combination of SNP arrays, CGH arrays, and targeted gene panel NGS data. CDK4 amplification and RB1 homozygous deletion were assessed in a subset of tumours. CDKN2A HD was associated with a dismal outcome in IDH-mutant astrocytomas (p < 0.0001 for PFS and p = 0.004 for OS) in both univariate and multivariate analyses. They suggested that IDH-mutant astrocytomas with CDKN2A HD should be considered grade 4 tumours irrespective of the presence of microvascular proliferation or necrosis [CDK4 and RB1 alterations did not correlate with clinical outcomes.To compare the utility of necrosis . InteresCDKN2A could stratify IDH-mutant astrocytomas, either alone or in combination with other molecular alterations [CDKN2A HD and survival in IDH-mutant glioma [CDKN2A HD as a predictor of significantly shorter PFS and OS in both LGG and glioblastoma in all included studies. Although this analysis included both astrocytomas and oligodendrogliomas, the authors noted that of the three studies reporting 1p/19q co-deletions, when the co-deletion was excluded, CDKN2A retained its prognostic value [Further reports using methylation data and FISH later emerged, indicating erations ,68,69. HCDKN2A/B in IDH-mutant astrocytomas. Roy et al. analysed the 9p region lost in malignancies by analysing two cohorts  and reported that CDKN2A inactivation did not promote tumour aggressiveness. Even when accounting for IDH and 1p/19q status (IDH-mutant 1p/19q non-deleted astrocytoma), there was no survival impact of CKDN2A HD. While they did show that heterozygous loss was associated with poor OS, mRNA expression was not altered. It was therefore postulated that this survival impact was due to the loss of other 9p genes [CDKN2A/B HD as a prognostic marker in IDH-mutant astrocytomas. Not all studies supported the use of 9p genes . It is uCDKN2A/B HD. They described a Japanese cohort of 308 low-grade gliomas that were comprehensively profiled for glioma-relevant genes via WES and CDKN2A/B via SNP array. The results were validated using a dataset of 414 LGG cases available from TCGA. Although alterations in retinoblastoma pathway genes  correlated with poor OS in general, CDKN2A HD alone did not correlate with OS in IDH-mutant astrocytomas  or IDH-wildtype astrocytomas  [Aoki et al. also failed to demonstrate the prognostic value of djusted) .CDKN2A/B on IDH-mutant astrocytoma prognosis. Satomi et al., using a combination of FISH and MLPA (multiplex ligation-dependent probe amplification), found a negative correlation between CDKN2A HD and OS in IDH-mutant grade 3 astrocytomas (n = 4/35 patients had a CDKN2A HD) (p < 0.001) but not in IDH-mutant high-grade gliomas (n = 13/27 had a CDKN2A HD) (p = 0.128). Astrocytoma was diagnosed with either the absence of 1p/19q co-deletion or loss of ATRX expression and strong diffuse p53 positivity. The non-significant finding in IDH-mutant glioblastoma and OS may be due to the small sample size [IDH-mutant astrocytomas for CDKN2A HD. They reported CDKN2A/B as a predictor of survival in morphologically grade 4 IDH-astrocytomas but not for grade 2 and 3 IDH-mutant astrocytomas [Other studies reported a more mixed effect of ple size . Anotherocytomas . The autIDH-mutant astrocytomas with CDKN2A/B HD, and reports related to their management are scarce. Reflecting this ambiguity, the current joint American Society of Clinical Oncology and Society of Neuro-Oncology guidelines recommend grade 4 astrocytomas be treated with concurrent temozolomide-radiotherapy with sequential temozolomide or radiotherapy alone with sequential temozolomide [There is no clear consensus on the treatment of zolomide . CDKN2A/B HD alters tumour biology (increased angiogenesis and cell growth), we cannot assume that these tumours will be as susceptible to temozolomide as their non-deleted counterparts. Unfortunately, the evidence for treatment specifically for CDKN2A/B HD astrocytomas is minimal. In 2000, Iwadate et al. investigated the relationship between CDKN2A deletion, p16 expression, and chemosensitivity to 30 different cytotoxic agents in vitro. They analysed 56 astrocytoma specimens  and found 17 specimens had p16 alterations . When looking at samples with p16 alterations, they found that deletions correlated with increased sensitivity to anti-metabolite agents but not to alkylating agents, antibiotics, topoisomerase inhibitors, or anti-microtubule agents [However, given the evidence that e agents . IDH-wildtype glioblastomas in patients before and after the use of bevacizumab. They were able to show that in the historical pre-bevacizumab group, there was a statistical difference in OS favouring the CDKN2A non-deleted group . However, the significance is lost in the bevacizumab-treated group . They proposed that the addition of bevacizumab should be considered in patients with CDKN2A HD glioblastoma [CDKN2A/B HD changes tumour response to treatment, given that they did not look at IDH-mutant tumours, it is hard to put them into the context of the current classification.Funakoshi et al. analysed OS in blastoma . While tCDKN2A/B homozygous deletions as a negative prognostic feature. As demonstrated in our literature summary in CDKN2B deletions in the absence of CDKN2A deletions. Overall, the literature supports the use of CDKN2A/B HD. For example, Cimino et al. proposed different grading systems based on a combination of CDKN2A/B HD and other molecular markers [CDKN2A/B alone as a molecular marker for grade 4 IDH-mutant astrocytoma, determined by a number of methods, including methylation, SNP array, and NGS [CDKN2A/B had no impact on survival in IDH-mutant astrocytomas; however, both studies used FISH [CDKN2A/B HD are prognostic in IDH-mutant astrocytomas, given the variety of testing methods used [The clinical studies in this review span five different CNS tumour grading classifications . This means that extrapolating most of the available evidence to the current clinical situation is difficult. Varying tumour classifications have been used to describe the effect of  markers ,19. Howe and NGS ,12,13. Ased FISH ,31. The ods used . CDKN2A/B heterozygous deletions impact survival. Roy et al. demonstrated that CDKN2A/B HeD did not impact survival and that survival differences seen in this group are likely due to deletions of surrounding genes [CDKN2A/B deletion may be related to the loss of the surrounding genes . The literature has not shown that ng genes  ThereforCDKN2A/B, this may also indicate the importance of these bystander genes in outcomes for astrocytomas with CDKN2A/B deletions [It should be noted that point mutations were also highlighted by some of the presented research. However, the evidence for this particular alteration is very limited and does not show an impact on survival. Again, as these alterations do not result in the loss of genes neighbouring eletions ,71.CDKN2B deletions. In the post-molecular era, 13 studies examined CDKN2A deletion, with only three looking at CDKN2B. There is a general assumption that, due to its close proximity to and similar function to CDKN2A, the prognostic effect of CDKN2B is likely similar. Supporting this assumption are early translational studies that demonstrated that CDKN2B deletions, in addition to CDKN2A, are related to the aggressive phenotype [CDKN2B HD in astrocytoma patients.There are also studies lacking a focus on henotype . HoweverCDKN2A/B HD IDH-mutant astrocytomas. The new entity of grade 4 astrocytoma with CDKN2A/B HD (WHO 2021) encompasses IDH-mutant astrocytomas previously treated with different regimens. Those previously classified as morphologic grade 3 astrocytomas would have been treated with radiotherapy alone followed by chemotherapy, while those previously characterised as IDH-mutant glioblastomas would have been treated with concurrent temozolomide-radiotherapy followed by sequential temozolomide. Therefore, which regimen should be used in IDH-mutant astrocytomas with CDKN2A/B HD is debatable. There is also a paucity of data on the management of CDKN2A/B HD have a direct oncogenic effect through loss of cell cycle inhibition and other parallel processes and are a molecular marker that influences grading and survival in IDH-mutant astrocytomas. Here, we review the evidence concerning CDKN2A/B deletions in a historical context. Overall, the evidence supports the use of CDKN2A/B HD as a negative prognostic marker in IDH-mutant astrocytomas. However, there is a significant variation in certainty, methods used for deletion detection, and the quality of the presented literature. There are also inaccuracies resulting from misclassification of tumours in older studies based on the revised WHO classification. These limitations hamper conclusions regarding the certainty and depth of impact CDKN2A/B HD has on prognosis and management and how this impact is affected by other co-occurring molecular alterations. Therefore, the strongest evidence for CDKN2A/B HD in IDH-mutant astrocytomas must come from prospective reports with the current WHO 2021 classification. Furthermore, clinical trials that use the current WHO 2021 classification are required to determine the optimal management of IDH-mutant astrocytomas with CDKN2A/B HD.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo–electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery. A spike antigen is designed with high expression that elicits a nAb response in mice effective against the Wuhan strain and VoCs. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China before spreading globally and being declared a pandemic in March 2020. With more than 613 million confirmed cases globally, resulting in more than 6.5 million deaths, COVID-19 remains a notable global health burden. Vaccines, such as mRNA-1273 (Moderna), BNT162b2 (Pfizer/BioNTech), and Ad.26.COV2.S (Janssen), are all based on the sequence of the original Wuhan-Hu-1 strain of the spike fusion glycoprotein (S), engineered to remain in the prefusion state, which is the primary target of neutralizing antibodies (nAbs). However, the efficacies of these first-generation vaccines are diminished against newly circulating variants of concern (VoCs) such as Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron , which evade nAbs due to mutations in the S protein , which are located between the heptad repeat region 1 (HR1) and the central helix (CH) domain  structures of our top design, S2D14, confirm the structural integrity of the spike protein and the dynamic nature of the RBDs, thus allowing exposure of potently neutralizing epitopes. This work provides a computational and experimental workflow for improving antigen characteristics, such as expression and immunogenicity, and may be valuable for informing the design of future spike-based vaccines that can elicit broad protection against emerging and future strains of the virus.A to D), which we obtained from the BLAST database (data S1)    293 cells. Among the 15 designs selected, 7 (three for the NTD + S2 and four for the S2 domain only) displayed expression levels in cell culture supernatants that exceeded S-2P, with five of those designs  also having expression levels in supernatant higher than S-6P . SpecifiKd values) in the picomolar range  was used to determine whether incorporated mutations within the NTD or S2 domain inadvertently altered the presentation or accessibility of key RBD epitopes. CR3022, which requires at least two RBDs in the open state, S309, which recognizes open or closed RBDs, and the hACE2 receptor, which binds a single RBD in the open conformation, were all found to bind designs 8, 9, and 10 (NTD + S2 domain) as well as designs 12, 13, and 14 (S2 domain) with equilibrium dissociation binding constants  VRC-118 and VRC-112, which target the NTD and S2 domains, respectively, was tested as these domains have also been shown to contribute to neutralization breadth and potency (m1 and Tm2) with an increase in Tm1 being indicative of improved stability , with design 9 having the largest, yet modest, increase of 4.2°C (Tm1 = 48°C)  were comparable to S-2P. The Tm2 values for most designs were also comparable to S-2P, with only designs 12 and 15 showing an ~10°C increase in melting temperature, though the impact of this improvement is not clear.To assess the impact of thermal stress, purified proteins were subjected to a differential scanning fluorimetry (DSF) assay. S-2P has previously been shown to have two distinct melting transitions (T = 48°C) . Tm1 valConsidering the loss of binding to VRC-112 for each of the designs, we next sought to determine whether modifications had inadvertently altered the S2 domain architecture or overall protein morphology. To accomplish this task, a Glacios 200-kV electron microscope was used to quickly assess one construct from the NTD + S2 designs and one construct from the S2 domain designs. Design 9 was chosen from the NTD + S2 designs based on the high expression and thermostability; however, particles resembling the prefusion S trimer were infrequently observed on the cryo-EM micrographs (fig. S4A). This may be due to dissociation into monomers or other misfolded states during storage or vitrification, consistent with known issues of cold handling with spike proteins  classification: one conformation with all three RBDs in the closed state and the second conformation with a single RBD domain in the open state  adjuvanted proteins at either 0.3- or 3.0-μg doses. Intramuscular injections were given on days 0 and 21, with serum collected 3 weeks post-I (first immunization) injection and two weeks post-II (second immunization) injection . Total aS2D14 elicited a robust neutralizing antibody response to the Wuhan strain at post-II for each vaccine dose . At postNext, we determined neutralizing antibody titers against VoCs that were available at the time of this study: Alpha (B1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (BA.1) strains . S2D14 eTo better understand the molecular basis for the improved breadth and immunogenicity of S2D14, a more extensive cryo-EM dataset was collected for high-resolution structural analysis using a Titan Krios 300-kV electron microscope. From a single dataset, we were able to determine the structure of S2D14 in three distinct conformations: two RBDs open , three RBDs closed , and two RBDs exposed with one RBD down  . TogetheTo better assess the relative proportion of RBDs in the open conformation, we performed BLI experiments measuring the peak binding levels for hACE2 and CR3022 to S-2P, S-6P, S2D14, and the S-2P–D614G variant. In this assay, an increase in peak binding signal indicates greater accessibility of the RBDs   present in S2D14 are all well resolved with clear side chain densities . Analysiα atoms (fig. S10). Despite this high structural similarity, there is a marked difference in the positioning of the loop connecting the α10 and α11 helices (residues G885 to Q895) near mutations A1070T and E1072Y located on the adjacent S protomer. These mutations reduce the overall charge of the interface between these two S protomers, creating a more hydrophobic patch that displaces the loop connecting α10 and α11 by 7.2 Å (measured from the Cα atom of residue A890) (Superposition of the S2 domain (residues 821 to 1147) of S2D14 in the two-RBD open conformation with S-2P reveals a root mean square deviation of 1.6 Å over all CThe emergence of SARS-CoV-2 variants has heightened concerns about the efficacy of currently approved vaccines, which are based on the prefusion stabilized S protein antigen, S-2P . A recent report of the immunogenicity of recombinant VSV vectored S-6P found that the enhanced stability and expression of S-6P led to the elicitation of a more effective nAb response at lower doses than S-2P against the Wuhan strain and VoC including Alpha (B.1.1.7), Gamma (P.1), Beta (B.1.351), Epsilon (B.1.427), and Delta (B.1.617.2) strains (Rosetta comparative modeling (RosettaCM) . Harvested cell media were centrifuged and cell supernatants were prepared in a 96-well plate with S-2P or S-6P standards diluted in media from 20 to 0.3125 μg/ml. The standard and mutant binding curves were measured using anti-polyhistidine biosensors (anti-HIS), where the concentration of each mutant in media was calculated by fitting the measured initial binding rate to the calibration curve. The expression levels were measured in duplicate wells of each mutant’s media and the average readout was reported.The culture supernatant of selected SARS-CoV-2 S mutants produced in a 1-liter scale was directly loaded through 5 ml of nickel–nitrilotriacetic acid Excel column (Cytiva Life Sciences). The column was washed with 300 mM sodium chloride and 50 mM imidazole in 20 mM Hepes buffer (pH 7.5) and captured S mutants were eluted with 300 mM sodium chloride and 250 mM imidazole in 20 mM Hepes buffer (pH 7.5). The collected samples from appropriate elution fractions were pooled and concentrated before further purification of the S trimer by gel filtration using a Superose 6 increase 10/300 GL column (Cytiva Life Sciences) with 1× phosphate-buffered saline (PBS), pH 7.4, as a running buffer. The fractions corresponding to the targeted S mutants based on SDS–polyacrylamide gel electrophoresis analysis were pooled together and quantitated using the absorbance at 280 nm.SPR experiments were performed in a running buffer composed of 0.01 M Hepes (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20 at 25°C using the Biacore 8K , with a series S protein A sensor chip . The ACE2 receptor or SARS-CoV-2 spike-specific antibodies (CR3022 or S309) were immobilized on the protein A sensor chip  at a ligand capture level of ~100 RU. Serial dilutions of purified spike designs were injected, at concentrations ranging from 10 to 1.25 nM. The resulting data were fit to a 1:1 binding model using the Biacore Evaluation Software .Nano-DSF was used to assess the thermal stability of purified spike designs on a Prometheus NT.48 instrument (NanoTemper Technologies). Samples were diluted to 0.2 mg/ml with PBS and 20 μl of each sample was loaded into capillary tubes. A temperature ramp was set to 1°C/minute, ranging from 20° to 95°C. The reported values are the mean of the first derivative of the ratio of intrinsic tryptophan fluorescence emission wavelengths for protein unfolding/folding (350 nm/330 nm), measured in triplicate.Octet binding assays were performed using the Octet RH96 system (Sartorius). Freshly purified S-2P, S-2P–D614G, S2D14, and S-6P at 10 μg/ml were loaded onto 16 HIS1K biosensors before being dipped into 200 nM CR3022 mAb or hACE2 . Immobilized antigen was allowed to associate with hACE2 or CR3022 for 10 min to achieve a signal plateau before a 10-min dissociation phase. The maximum binding signal of CR3022 and hACE2 was measured in triplicate.N = 10 mice per group) with AS03-adjuvanted  spike proteins at two dosage levels of 3.0 or 0.3 μg (N = 4) but was not considered for statistical analysis. Spike proteins and AS03 were admixed shortly before injection. Mice were injected intramuscularly twice 3 weeks apart and bled 3 weeks after the initial immunization (post-I) and 2 weeks after the second immunization (post-II).An in vivo study was performed to assess the immunogenicity of S2D14 compared to S-2P in a mouse model. Female BALB/c mice, 7 to 8 weeks of age at the start of the study, were immunized  Guidelines as applicable and were conducted in compliance with provisions of the United States Department of Agriculture Animal Welfare Act, the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the U.S. Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals.n = 22) were obtained at CHU Tivoli, Belgium, from donors 23 to 61 years of age, mostly from females, after polymerase chain reaction–confirmed diagnosis and at least 28 days after the participants were asymptomatic. Human samples were obtained with informed consent. All recruitment, sample collection, and experimental procedures using human samples have been approved by relevant institutional review boards and by GSK human sample management board. nAbs in post-II serum samples from mice immunized with S-2P, and S2D14 or placebo were additionally measured using a pseudo-virus neutralization assay with the Alpha, Beta, Delta, and Omicron variant strains .The serum CoV2-specific antibody responses were assessed using a Wuhan strain pseudo-virus neutralization assay to measure functional antibodies as described previously  model for repeated measures was fitted on logGeometric mean titers (GMTs) with corresponding 95% confidence intervals and geometric mean ratios (GMRs) with 90% confidence intervals  were computed from these models to compare responses to S-2P and S2D14 vaccines by dose. GMRs for which the confidence interval does not include 1 are considered statistically significant. The analyses included more vaccination groups than reported  as the in vivo study initially consisted of additional vaccinated groups irrelevant to this study, but a multiplicity of comparisons was not considered. GMTs with corresponding 95% confidence intervals for HCS samples were computed separately from vaccination groups using Prism .SARS-CoV-2 S designs 9 and S2D14, previously isolated by nickel-affinity chromatography, were further purified by size exclusion chromatography using a Superose 6 10/300 GL column (Cytiva Life Sciences) with tris-buffered saline buffer composed of 10 mM tris (pH 7.5) and 150 mM NaCl. Fractions containing purified spike protein were diluted to a final concentration of 0.4 mg/ml before specimen preparation on EM grids. Prepared samples (3.0 μl) were then applied to glow-discharged Quantifoil 1.2/1.3-400 mesh copper grids, blotted, and plunged into liquid ethane using an FEI Vitrobot Mark IV vitrification apparatus set at 100% relative humidity and 4°C (Thermo Fisher Scientific).A total of 1425 movies were collected using an FEI Glacios TEM at 200 kV equipped with a Falcon 3 direct electron detector at a magnification of ×120,000 corresponding to a pixel size of 0.91 Å/pixel. A more detailed description of imaging parameters used during data collection can be found in Cryo-EM single-particle analysis was carried out using the RELION 3.1 image processing suite  criteria.A total of 6169 movies were collected using an FEI Titan Krios operating at 300 kV and equipped with a Falcon 4 direct electron detector  at a magnification of ×120,000 corresponding to a pixel size of 0.67 Å/pixel. A more detailed description of settings used for imaging during data collection can be found in Cryo-EM single-particle analysis was carried out using the RELION 3.1 image processing suite (Modeling of each cryo-EM structure was performed using Phenix and COOT (