Thermal degradation of nylon-6 tennis string nylon wastes in inert nitrogen and air atmospheres was investigated by means of multiple heating-rate thermogravimetric analyses. The results obtained under the heating rates of 5–20 K/min are compared in terms of degradation feature and specific temperature for two atmospheres. Using nonisothermal data, kinetic analysis was thoroughly conducted using various isoconversional model-free methods, including Starink, Madhusudanan–Krishnan–Ninan, Tang, Coats–Redfern, and Flynn–Wall–Ozawa methods. With these kinetic analysis methods, the activation energy over the entire degradation process was successfully calculated. By means of the model-fitting master-plots method, the first-order chemical reaction model was determined to be the most appropriate mechanism function for describing pyrolysis and oxidative thermal degradation of nylon-6 waste. Using kinetic parameters, satisfactory matching against experimental data resulted using the Coats–Redfern method for both cases. Furthermore, thermodynamic parameters such as changes in entropy, enthalpy, and Gibbs free energy during thermal degradation processes were evaluated. Today, tennis is a very popular sport all around the world and tennis balls, rackets, and strings along with other auxiliary supplies are drastically consumed. However, tennis produces various wastes such as tennis balls, rackets, and strings in considerable amounts as they are readily worn out under high energy and high-speed shots during tennis. Among various tennis string materials, nylon-6 is the most commonly used polymer in order to achieve the best performance in tennis playing. Nylon-6 is one kind of polyamide characterized by the amide group (–NHCO–) and it is one of the most widely used engineering plastic in many industrial fields including building, home textile, automotive and electrical industries since it possesses good chemical stability, high mechanical strength, and elasticity. Furthermore, it is extensively used to fabricate tennis string because of its excellent wear resistance meeting the requirements of tennis sport to a great extent.cis-elimination or intramolecular rearrangement of amide groups and more aromatics and light unsaturated hydrocarbons will be yielded when using greater amounts of zeolite. Interestingly, Pannase et al. [In the present work, nylon-6 tennis string wastes in Tianjin, China, were collected for inert and oxidative thermal degradation considerations. It is well-known that pyrolysis and combustion are two effective methods to chemically converse plastic wastes into valuable substances for sustainable energy development ,2. To dae et al.  reportedaE, pre-exponential factor A and reaction mechanism model function f(α) using isothermal thermogravimetric (TG) data or dynamic non-isothermal TG data [Ea calculated at 190 kJ/mol along with the first-order reaction for pyrolysis of fresh nylon. Similarly, Bockhorn et al. [Ea of 211 kJ/mol, logA of 15.0 min−1 and a reaction order of 0.81 for nylon-6 over the dynamic degradation process while in the case of isothermal condition nylon-6 had a lower Ea of 205 kJ/mol, lower logA of 14.5 min−1, and higher reaction order of around 1.0. As a comparison, the Ea was greatly reduced to 113 kJ/mol using a basic catalyst of NaOH/KOH eutectic mixture, along with log A = 8.7 min−1 and a reaction order of n = 1.06. However, with an acid catalyst of H3PO4, the Ea was also reduced to Ea = 164 kJ/mol, together with log A = 14.0 min−1 and a reaction order of n = 1.9. A rather similar Ea value of 205 kJ/mol has also been reported in the work of Pannase et al. [Apart from the study of degradation product compositions, kinetic analysis of thermal degradation of nylon-6 has also been attempted in terms of kinetic triplet parameters of as apparent activation energy  TG data ,7,11,12. TG data  studied aE of 266 and 220 kJ/mol for fresh fishing nets and waste fishing nets, respectively. They also reported the reaction order of 0 or 1 for fresh nylon and waste nylon, respectively. Likewise, Eimontas et al. [Furthermore, kinetic analysis studies for pyrolysis of nylon-6 wastes have also been conducted and can be referred to in two works ,7. Kim eEa, lnA, and f(α) with a number of isoconversional kinetic analysis methods and the calculation results with these methods are discussed in detail. The variation in activation energy with conversion can be used to reveal the complex nature of the thermal degradation reaction. (3) The model-fitting master-plots method has been attempted to scan the most appropriate mechanism model for describing oxidative or inert thermal degradation processes of nylon-6 tennis string wastes and the first-order chemical reaction model is found to be obeyed during thermal degradation processes. With these kinetic parameters, rebuilding the mass conversion curves has been performed, consequently resulting in satisfactory predictions against experimental results. (4) Finally, thermodynamic functions such as changes in entropy, enthalpy, and Gibbs free energy during pyrolysis degradation have also been quantified by assuming the transition state theory. The current work provides detailed information about thermal degradation behaviors and kinetic triplet parameters necessary for designing a chemical reactor to thermally dispose of nylon-6 wastes.The objectives of the present work are: (1) thermal characteristics and degradation kinetics of nylon-6 string wastes under inert nitrogen and oxidative air atmospheres are compared by means of thermogravimetric analysis. (2) The non-isothermal mass loss data obtained are kinetically analyzed to determine the kinetic parameters of ®Taan of Shenzhen Taiang Industrial CO., Ltd., Shenzhen, China) was collected locally in Tianjin, China. The collected filaments were washed using distilled water and sun-dried in open air. Then, the dried sample after cutting was milled with a grinder and meshed to less than 100 um powder. The resultant powder was dried at 400 K in a controlled oven for 20 h to remove moisture residual and finally kept in an airtight container for subsequent measurements. Tennis string waste, from the same brand  for thermal degradation measurements and differential thermal analysis (DTA) under a constant heating rate mode. The purging gas used was air or N−1. FTIR results obtained with 32 scans for each run can be used to identify the chemical structure of real-world nylon-6 waste samples. The powder waste sample after being further milled with a mortar and pestle was mixed with KBr at a mass ratio of 5:95 and then pressed into a transparent sample disc for subsequent FTIR measurements.Fourier transform infrared (FTIR) analysis was performed on nylon-6 tennis filament waste samples via a Bruker Alpha-H infrared spectrophotometer and FTIR data acquired varied in the wavelength range of 400–4000 cmEa), the pre-exponential factor (A), and the reaction mechanism function (f(α)) [Ea values are calculated as a function of the degree of mass conversion (α). Using the model-fitting method, the most appropriate reaction mechanism function (f(α)) can be properly found based on the Ea values, and the third kinetic parameter A can be readily determined thereafter. With these kinetic parameters, thermal degradation TG and DTG curves can be predicted and necessary information for subsequent industrial applications can be provided. Therefore, model-free and model-fitting methods are both attempted for analyzing thermal degradation of tennis string waste.Deep analysis of thermal degradation kinetics is a very useful tool for securing the thermal degradation process and explore degradation reaction mechanisms. Theoretical kinetics analysis has been commonly performed using isoconversional model-free and model fitting methods and the thermal degradation of various solids can be accurately characterized by using kinetic triplets of the activation energy (n (f(α)) ,14. Withdα/dt) as a function of time and temperature given below:k(T) is the rate constant of thermal degradation reaction and is usually written as the Arrhenius equation k(T) = A·exp(-Ea/RT), in which aE(J/mol) is the activation energy to satisfy degradation reaction. A (min−1) is the pre-exponential factor, T (K) is the absolute temperature and R is the universal gas constant (8.314 J/mol K). Furthermore, f(α) stands for a reaction mechanism function for describing the kinetic thermal degradation, and α is set to be the extent of mass conversion defined as: 0m, tm, and m∞ are the initial sample mass, the sample mass at degradation time t, and the final sample mass, respectively. So far, the isoconversional analysis method has been the most commonly used for thermal degradation kinetics and then non-isothermal temperature program is conducted at a constant heating rate mode with β defined as β = dT/dt. Subsequently, Equation (1) can be transformed as: Generally, thermal reaction process of plastic solids may be described by the progress of degradation (g(α), can be deduced from Equation (3) by rearrangement and integration, given as:Readily, an integral form of reaction mechanism function, f(α) and integral form of g(α) are available [As for describing the thermal degradation mechanism, a number of reaction model functions in differential form of vailable  and somex = aE/RT and In Equation (4), a well-known temperature integral function is involved and can be further transformed by setting p(x), and thus numerous approximate solutions or numerical regressions have been proposed such as Starink (SK) [However, there is no analytical answer to solve ink (SK) , Tang etink (SK) , Madhusuink (SK) , Coats–Rink (SK) , and Flyink (SK) ,20. TheyEa can be easily evaluated over the whole conversion range. As such, these methods are thought of as model-free methods since they do not consider any specific chemical reaction model. Currently, these integral methods have been extensively used to analyze non-isothermal TG data and then isoconversionally calculate activation energy at any arbitrary mass conversion level ,23,24,25Ea but they are not directly related to the identification of the thermal degradation model. On the contrary, the model-fitting method has been frequently applied for such an aim. Among various model-fitting methods, one of the most frequently reported is the master-plots method given by Gotor et al. [g(α) at α = 0.5:0.5x equals aE/0.5RT and T0.5 is a specific temperature set at α = 0.5. Based on Equations (5) and (11), a mathematical expression to represent the most common master-plots method [g(α)/g(0.5) and p(x)/p(x0.5) curves are condensed to 1 at α = 0.5. By respectively drawing experimental p(x)/p(x0.5) values versus α and theoretical g(α)/g(0.5) values versus α of various reaction models listed in As we can see, the above-given model-free methods can rather conveniently result in mass conversion dependent r et al.  to appro α = 0.5:g(0.5)=AEs method ,22,26, nEa), changes in enthalpy (ΔH), changes in entropy (ΔS), and changes in Gibbs free energy (ΔG), were calculated based on transition-state theory using the following expressions [e is the Neper number (2.7183), h and kB represent the Plank constant (6.626 × 10−34 J/s) and Boltzmann constant (1.381 × 10−23 J/K), respectively. Herein, A is the pre-exponential factor determined as above and Ea the activation energy from the SK method.Thermodynamic quantities for pyrolysis and oxidative thermal degradation of tennis string nylon-6 waste, including activation energy −1 can be attributed to the stretching vibrations of hydrogen-bonded NH groups while the adsorption bands centered at 3072, 2912, and 2843 cm−1 can be assigned to the stretching vibration of aliphatic CH groups for the nylon-6 waste sample. Meanwhile, the absorption peak at 1623 cm−1 can be accounted for as the stretching vibration of amide II and CN groups of nylon-6 and the one at 1539 cm−1 may be due to the vibrations of amide I [2 and programmed from ambient to 850 K under four ramping rates of β = 5, 10, 15, and 20 K/min. Clearly, as found from the TG results, the mass-T curve has moved to higher temperature ranges upon enlarging the heating rate β and the temperature movement is applied to both cases. This result may be attributed to a combined effect of different heat transfer and distinct kinetic rates, which in turn, lead to the reduced gasification rate of the reaction product with the rise in heating rate and subsequently a temperature-shift compensation.2 than in oxidative air, but in both cases, the waste sample has completely burned out without any visible solid residual. As can be seen, nylon-6 begins to degrade in oxidative air when heated upwards to 581 or 622 K upon employing different heating rates, and its complete degradation may be accomplished when heating the sample up to 737 K under 5 K/min or 770 K for the case of 20 K/min. In contrast, the pyrolysis in inert N2 for nylon-6 string wastes is seen to undergo in-between 606 to 742 K or from 643 to 772 K when augmenting β from 5 K/min to 20 K/min. With these TG data, kinetics analysis can be performed for inert and oxidative thermal degradation processes and will be discussed later. Similarly, Pannase et al. [A careful examination of e et al.  recentlye et al. ,10.2 and air atmospheres, and one can see that all DTG curves have displayed only one peak for every heating rate, at which the sample has reached a maximum mass loss rate (dα/dt) and the temperature at the peak is normally defined as Tp. Like those shown for the TG results, the DTG results are also significantly affected by the variation in β and DTG curves moving rightwards to high-temperature domains as β rises as reflected by Tp value is ascended and the peak turns wide and intense as β rises up from 5 to 20 K/min. These observations are the same for both cases of N2 and air. As seen from Tp values are unsurprisingly lower in air than in N2. Furthermore, one can deduce from these one-peak DTG results that the nylon-6 string wastes may have undergone one reaction stage during thermal degradation processes and thus a single-reaction model assumption may be somewhat reasonable for describing kinetic degradations according to ICTAC Kinetics Committee recommendations [ndations . Such anT5, T15, and T60, and they are defined as the temperatures, respectively, at 5% mass conversion (T5), 15% mass conversion (T15), and 60% mass conversion (T60). For both cases of inert and oxidative atmospheres, each of these specific temperatures is linearly promoted with the increase in the heating rate and a linear expression may be derived for quantitatively describing the heating-rate dependence of each specific temperature. As can be seen from 6 demonstrated rather better thermal stability in N2 than in air [Looking at n in air . 2, the crystal in the nylon-5 waste sample melts at 498, 500, 502, and 504 K when heated at 5, 10, 15, and 20 K/min. In the case of air atmosphere, the melting points become relatively lower as compared to the inert case, similar to those TG data discussed above. These melting point values are consistent with those in the literature [During pyrolysis, the nylon-6 tennis string waste sample experienced two heat response stages—endothermic melting and exothermic decomposition, respectively. terature ,32 and cterature ,32.β/T1.92) against 1000/T for the nylon-6 degradation in both air and N2 are presented in α-dependent aE values calculated from the slope of each Arrhenius plot are listed in R2 values are also listed in β/1.92T) on 1/T for each conversion level.With the SK method, the plots of ln vs. 1000/T over 0.05 < α < 0.95 are given in 2 pyrolysis processes. Similar to the SK method, a very good linear relationship can be observed between ln(β/T1.896441) and 1/T, and the R2 values can be referred to in aE values can be estimated using Equation (7) and the calculated results for all α values are presented in aE data in aE value computed by using the Tang method shows the same trend as that resulted from the SK method. For the case of oxidative degradation, the activation energy aE value shows relatively large deviations and it varies from 90.53 to 178.20 kJ/mol for 0.05 < α < 0.95. In contrast, the aE value for N2 pyrolysis varies within from 142.84–207.99 kJ/mol. The averaged aE values are 151.09 and 188.98 kJmol−1 for air and N2 cases, respectively. Unsurprisingly, the deviation of the α-dependent aE from the averaged aE for both air and N2 cases exhibited a similar result to that observed for the SK method. Hence, the Tang method could be used for describing pyrolysis in N2 or oxidative thermal degradation regardless of relatively large activation energy fluctuations.The isoconversional Tang method is considered for analyzing thermal degradation kinetics of string wastes in both NaE value following Equation (8). Accordingly, the Arrhenius plots of ln(β/T1.884318) vs. 1000/T over 0.05 < α < 0.95 are also shown in aE values thus obtained are tabulated in aE values alter with the conversional extent in N2 with a relatively larger deviation than in air. In the case of N2, the aE value gains momentum from 142.82 to 188.95 kJ/mol as α increases and it varies within 90.54–178.17 kJ/mol for oxidative thermal degradation. The averaged aE over the entire mass loss range is 188.95 for N2 and 151.08 for air. Similar to the SK and Tang methods, the isoconversional MKN method has been employed to determine the β/2T) versus 1000/T over 0.05 < α < 0.95 for thermal degradation in air or N2 pyrolysis processes are presented in aE calculated from the slope of each plotted line according to Equation (9) and resultant R2 values are given in β/2T) and 1/T is very well as is also reflected by the R2 values listed in aE value relies intensely on mass conversion α and it spans from 90.10 to 177.83 kJ/mol for N2 pyrolysis while from 142.46 to 207.64 kJ/mol for oxidative thermal degradation. On average, the aE values thus obtained are 150.69 and 188.63 kJ/mol for air and N2 cases, respectively. Attempting the CR method for analyzing kinetics, the Arrhenius plots of ln(β) against 1000/T are depicted in 2, and the aE and R2 values thus calculated for each α are given in R2 values over the entire conversion range are found to closely approximate 1.00, showing a linear dependence of log(β) on 1/T for every conversion level. The aE values via the FWO method are known to be readily estimated from the line slopes of all Arrhenius plots shown in aE values for either N2 or air are strongly dependent on α and they vary from 95.82 to 180.42 kJ/mol for pyrolysis or from 145.91 to 209.15 kJ/mol for oxidative thermal degradation as α varies from 0.05–0.95. If averaged over the entire conversion range, the aE values thus calculated are 190.61 and 154.38 kJ/mol, respectively. Following the FWO method, the Arrhenius plots of log of nylon-6 waste has been identified by using the model fitting method. Thus, the G-master plots method has been applied here for this purpose [g(α)/g(0.5) vs. α against experimental plots of 5, 10, 15, and 20 K/min for both air and N2 cases. All the reaction functions presented in 2 case, only seven chemical reaction equations of F1/3, F1/2, F2/3, F3/4, F4/5, F9/10, and F1 have given rather better condensations on experimental plots for 0.05 < α < 0.50 whereas the other reaction models are observed to be further away from the experimental values. While for 0.50 < α < 0.95, five Avrami–Erofeev mechanism functions, D3, D7, F2, F3/2, F1/3, and P2 models are observed to lead to very large deviations against the experimental data, and D6, D8, F1/2, and P3/2 models are seen to result in certain deviations as compared to experimental values, the remaining five chemical order mechanism functions of F2/3, F3/4, F4/5, F9/10, and F1 are found to very well condense on experimental plots. Apart from obtaining the  purpose ,21,22,262 case is applied as α varies in the range of 0.05–0.50 as shown in α <0.95, the Avrami–Erofeev mechanism functions including A3/4, A3/2, A2, A5/2, and A3, along with D3, P2, F3/2, F2, F1/2, and F1/3 models, resulted in very large deviations against experimental curves while P3/2, D6, D7, and D8 models also led to minorly poor performance. Promisingly, the left F2/3, F3/4, F4/5, F9/10, and F1 reaction models are seen to very well approximate experimental plots. Due to the assumption of the one-stage reaction for the entire oxidative and pyrolysis thermal degradation processes, the F2/3, F3/4, F4/5, F9/10, and F1 reaction models can then be considered further as they perform very well over 0.05 < α <0.95, and their simulated results will be compared so as to identify the most suitable reaction models for thermal degradation of nylon-6 string waste. In the literature, the first-order reaction function for pyrolysis of nylon-6 and its waste has already been reported [In the case of oxidative thermal degradation, the same finding for the Nreported . Furtherreported  showed aexpα and cala are, respectively, the mass conversion experimentally obtained and calculated with the reaction model, and N is the total number of mass conversion considered here. 2 cases. In this sense, the F1 model can be readily thought of as the most appropriate reaction model for pyrolysis and oxidative thermal degradation of nylon-6 tennis string wastes. With the Ea and F1 model, the pre-exponential factor lnA values were then calculated according to Equation (8) and the results thus obtained—as a function of α—are listed in A results presented in A value varies from 12.50 to 29.03 for the air case, and from 22.13 to 34.11 for the N2 case, respectively. Accordingly, the A value ranges from 2.68 × 105 to 4.03 × 1012 min−1 and 4.08 × 109 to 6.51 × 1014 min−1 for the air and N2 cases, respectively.The comparison of the above-mentioned five models was conducted by fitting them against experimental data by the very commonly CR method in terms of the absolute average relative deviation (AARD) defined as:Ea and lnA, indicative of a compensatory increase in lnA with an increase in aE [Ea and lnA are kJ/mol and min−1, respectively.A further examination shows that there is a kinetic compensation effect between the se in Ea . Such a Ea and lnA parameters over the entire range of 0.05–0.95 performed satisfactorily to recast the temperature-dependent mass-conversion curves for nylon-6 waste samples since all the experimental data points selected are mostly condensed on their respective α-T curves. With fully obtained kinetic parameters, the theoretical predictions to thermal degradation processes can be made, and G, ΔH, and ΔS have been evaluated with the aid of the aE values resulting from the CR method and lnA from the F1 model, and the calculation results are listed in H value in oxidative air than in nitrogen over the entire conversion range, requiring less energy to degrade at any conversion level. All the ΔH values are positive in either air or N2, and such endothermic features mean oxidative and inert thermal degradations are all heat absorbed. As seen from G values are almost kept constant at 186.51 and 185.85 kJ/mol over 0.05 < α < 0.95 for nitrogen and air cases, respectively. Thermodynamically, all positive ΔG means non-spontaneous degradation processes in both atmospheres, and therefore, heat must be introduced to undergo thermal degradation. The higher the ΔG, the less favorable to undertake the degradation process. Finally, most ΔS values are negative during the degradation process as observed from H and ΔG values span from 84.71–171.85 kJ/mol and 185.40–186.41 kJ/mol for oxidative degradation, and 136.97–201.48 kJ/mol and 184.59–187.44 kJ/mol for N2 pyrolysis, respectively. Meanwhile, the ΔS values vary from −75.85 to 22.79 J/mol·K for pyrolysis or from −155.76 to −19.23 J/mol·K for oxidative thermal degradation. Similarly, Pannase et al. [H, ΔG, and ΔS values—198.89 kJ/mol, 199.72 kJ/mol, and −1.09 J/mol·K, respectively.Thermodynamic analysis of pyrolysis and oxidative thermal degradation processes for nylon-6 tennis string wastes can be accomplished based on the assumption of transition state theory ,24,28. Ae et al.  also rep2 and oxidative air atmospheres with TG measurements at a ramping rate of 5–20 K/min. Five integral methods—SK, Tang, MKN, CR, and FWO—were carried out to calculate activation energies over the entire conversion range. The master-plot method was used to search the most appropriate degradation model for describing pyrolysis and oxidative thermal degradation of nylon-6 waste. Some concluding remarks may be drawn from the present work:(1)2 than in air, meanwhile, the degradation thermal temperatures were found to linearly rise with the heating rate.Nylon-6 waste is more thermally stable in N(2)2 pyrolysis.For kinetically analyzing thermal degradation of nylon-6 waste, a one-stage reaction model is assumed for elaborating both non-isothermal thermo-oxidative degradation and N(3)aE values over 0.05 < α < 0.95, calculated by using the isoconversional SK method, vary from 95.82 to 180.42 kJ/mol for N2 pyrolysis or from 145.91 to 209.15 kJ/mol for oxidative thermal degradation, respectively. The variation in aE with respect to α suggests that the chemical reaction of varying extents of complexity may have occurred during pyrolysis or oxidative degradation.The (4)aE values while the lowest aE values were obtained by using the CR method, and the aE value over the entire conversion range follows the sequence: CR < SK < MKN < Tang < FWO for both atmospheres.Among five model-free methods, the FWO method yielded the highest (5)By means of the model-fitting G-master-plots method, many reaction models were evaluated with the most common CR method and the first-order reaction model was found to be the most suitable mechanism function for describing pyrolysis and oxidative degradation of nylon-6 waste—as proven by the lowest calculation deviations. The CR method together with kinetic triplet parameters rebuilt multiple α-T curves with satisfactory performances.(6)H, ΔS, and ΔG were estimated according to the transition state theory, and over the entire degradation process, the ΔH, ΔG, and ΔS values thus calculated varied from 137 to 201 kJ/mol, 185 to 187 kJ/mol, and −76 to 23 J/mol·K for N2 pyrolysis, or from 85 to 172 kJ/mol, 185 to 186 kJ/mol, and −156 to −19 J/mol·K for oxidative degradation, respectively. These results suggest that it is unfavorable for nylon-6 tennis string wastes to undergo thermal degradation and more energy is required for conducting pyrolysis than utilizing oxidative thermal degradation.Thermodynamically, ΔIn the present study, non-isothermal features and degradation kinetics of nylon-6 tennis string waste were studied under N


The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions  on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials. These products employ AAV1, AAV2, and AAV9, respectively, as vectors1. Moreover, AAV vectors have been successfully used in liver-targeted gene therapy, most notably for hemophilia5. However, AAV-based liver-directed gene therapies must overcome several challenges prior to their clinical applications6. A major hurdle is the poor efficacy associated with transduction into human hepatocytes as compared to that into mouse liver cells, caused by species-specific differences in AAV tropism between mice and non-human primates. Although AAV8-based vectors are approximately 10- to 100-fold more efficient than AAV2- or AAV5-based vectors in mouse liver transduction, accumulating preclinical and clinical data indicate that AAV8- and AAV5-based vectors, which are used in most current clinical protocols are not efficient at specifically targeting primary human hepatocytes. Further, the data from preclinical models are not predictive of their clinical performance in humans8.Adeno-associated virus (AAV) vector-mediated gene delivery has demonstrated promising therapeutic effects in many clinical trials. Currently three AAV gene therapy products, authorized by the US Food and Drug Administration and the European Medicine Agency, are available: Glybera  for lipoprotein lipase deficiency, Luxtruna (voretigene neparvovec-rzyl) for inherited retinal degeneration, and Zolgensma (onasemnogene abeparvovec-xioi) for spinal muscular atrophy9 and 1:1 for AAV-Spark100 (engineered capsid derived from AAVrh74)3, have been associated with reduced, or abrogated, therapeutic efficacy. Thus, the presence of NAbs against AAV capsids is a common exclusion criterion for participation in the clinical trials. The seroprevalence rates against wild type AAV in humans vary depending on various factors such as age, geographic location, and species of origin of the capsid, and can reach 30% for AAV5, 40% for AAV8, and 70% for AAV1 and AAV210. This substantially reduces the proportion of the population that could benefit from AAV- based therapeutics. Due to the high degree of conservation in the amino acid sequence among AAVs, cross-reactivity of anti-AAV antibodies between a wide range of serotypes is typically > 50%. Therefore, a substantially large proportion of the population cannot benefit from AAV-based therapeutics unless the vectors are designed to evade NAbs.Another hurdle facing liver-directed gene therapy is the high prevalence of neutralizing antibodies (NAbs), which diminishes the efficacy of AAV-based therapies after systemic administration. Even low titers, such as 1:17 for AAV211 and non-human primate7. Moreover, the novel synthetic liver-trophic capsid AAV-LK03 generated by a directed evolutional approach shares > 90% capsid homology with AAV312. Another liver-trophic vector, AAV3B-DE5, was recently reported by applying directed evolution specifically to AAV313. In this study, we employed a bioengineering process to rationally design the AAV3 capsid. Triple substitution of amino acids, S472A, S587A, N706A, on the surface loop of the AAV3B capsid protein was studied to assess their transduction efficiency into primary human hepatocytes and enhanced resistance to pre-existing human NAbs.It is, therefore, imperative to explore effective approaches for the enhancement of AAV transduction and simultaneous NAbs evasion. Recently, studies have shown that AAV3 vectors were able to efficiently transduce human hepatocytes in a humanized mouse model16, as well as antigenic epitope mapping of AAV1, 2, 5 and AAV817, we identified three variable regions  in the surface loops of VP3 as important for reducing the immunogenicity. We substituted three amino acids, one in each variable region considered to be involved in antibody binding, with alanine -expressing AAV vectors at a 104 multiplicity of infection (MOI) in HepG2 cells, the mean fluorescence intensity (MFI) of GFP (n = 4) were found to be 10.8 for AAV8, 79.8 for AAV2, 449.8 for AAV3B, and 485.3 for AAV.GT5. Normalization of the MFI with respect to that of AAV8 showed that the GFP expression levels caused by AAV2, AAV3B, and AAV.GT5 were 7, 42, and 45 times greater than that by AAV8, respectively  antibodies against AAV.GT5, AAV2, and AAV3B in 106 healthy adults. To capture AAV-specific IgG antibodies in an enzyme-linked immunosorbent assay (ELISA), each vector particle was immobilized on plates. NAb titer 1:32 corresponded to an optical density (OD) of 0.527 for AAV.GT5, 0.349 for AAV2, and 0.486 for AAV3B in the ELISA  Results from preclinical studies on animals do not necessarily translate directly into humans. Species or even strain differences in the transduction efficiency of AAV vectors have led to disappointing results in the development of vectors. For example, engineered capsid AAV.PHB.B, which exhibited broad transduction in the brain of some mouse strains, was found to not be so effective in other strains of mice and non-human primates21. (2) The high prevalence of NAbs to AAV capsids hinders systemic administration of the vectors24. Although successful liver transduction was reported with AAV5 vector in the presence of pre-existing anti-AAV5 NAb titers up to 1:1,030 for non-human primates and 1:340 for humans25, it remains to be elucidated if this evasion from NAbs is specific for AAV5 vectors. (3) Transgene expression declines following elimination of transduced hepatocytes26. (4) High doses of the vectors are more likely to elicit a host immune response. Possible dose-dependent toxicity of AAV vectors has recently been highlighted by the deaths of three patients in a clinical trial for the X-linked myotubular dystrophy in which 3 × 1014 vg/kg of AAV8 vectors was systemically administered28. Therefore, it is imperative to explore effective approaches for the enhancement of AAV transduction and NAb evasion.The liver is a preferred target for gene therapy against several genetic and metabolic disorders14 and AAV3B15. These isolates differ in eight amino acids of capsid proteins, although functional differences between the two isolates have not been elucidated. We selected AAV3B as the parent AAV for capsid modification, since most of the previous reports on AAV3 used AAV3B. AAV3 uses the human hepatocyte growth factor (HGF) receptor as a co-receptor for cellular entry29 and has been shown to efficiently transduce human hepatocytes30. Recent structural analysis by X-ray crystallography and cryo-electron microscopy on various AAVs suggested that the surface topology of capsid proteins are conserved across all AAVs31. Nine common variable regions (VR-I to VR-IX) were identified among AAV2, AAV8, AAVrh8, and AAV916. Differences in the surface loops of these VRs affect receptor binding, transduction efficiency, and antigenicity. Possible epitopes of antibodies against AAV capsids have been mapped using various approaches including peptide scanning, peptide insertion, and site-directed mutagenesis17. For example, epitopes of AAV2 and the footprint of C37-B antibody include residues 492–498 on VR-V and 585–589 on VR-VIII31, while peptide scanning using D3 antibody identified residues 474–483 on VR-IV32, and site-directed mutagenesis mapped multiple residues including 263, 264, 384, 385, 548, and 708 for A20 antibody33. Since the capsid VP1 proteins from AAV2 and AAV3B were 83% identical15, we predicted that NAbs that cross-react with AAV2 and AAV3B also recognize these residues.In this study, we engineered AAV3. There are two isolates of wild type AAV3, AAV3A34. E563A substitution also eliminated capsid protease activity35. With Y704A substitution, second-strand synthesis and transcription were impaired36. It is also important not to interfere with the function of assembly activating protein (AAP), which is translated across the start codon of VP337. Although the binding site of HGF on the AAV3 capsid has not been elucidated, residue 594 has been identified as a binding site for heparan sulfate proteoglycan (HSPG)38.Previous mutagenesis studies on the AAV2 capsid protein demonstrated negative effects after specific amino acid substitutions. With E563A and H526A substitutions, infectivity was severely reduced39, we confirmed that prevalence of AAV-specific IgG and NAb activity were reduced in AAV.GT5 with S472A, S587A, and N706A substitutions. Since addition of chaotropic agents such as guanidine thiocyanate or urea did not reduce the avidity of the AAV-specific IgG in AAV.GT5 compared with AAV3B, triple mutations may affect the accessibility of epitopes rather than the binding strength of the NAbs. Moreover, we observed efficient transduction of AAV.GT5 into primary human hepatocytes derived from chimeric mice with human liver (PXB cells), as well as into two human hepatocellular carcinoma cell lines. The transduction efficiency of AAV.GT5 was comparable with that of parental AAV3B, and significantly greater than that of AAV8, AAVrh37, and AAV-Spark100. Although PXB cells recapitulate many aspects of human hepatocytes, it remains unclear how well the human xenograft model could predict transduction efficiency in the human body40. As an approach to address this concern, we have recently initiated in vivo transduction studies on non-human primates comparing AAV.GT5 expressing human coagulation factor IX with other liver-directed AAV vectors.Based on these findings, we selected residues 472, 587, and 706 from VR-IV, VR-VIII, and VR-IX, respectively, for engineering the AAV3 capsid. These three residues are conserved between AAV3A and AAV3B, however are diverse in other AAVs. We used a conventional strategy by substituting the amino acids in these position with alanine, a small amino acid, to eliminate effective interactions between capsid proteins and NAbs. Although rational design is still challenging and substitution to alanine may enhance the antibody binding41. In the wild type AAV seropositive individuals, IgG1 appears to be the predominant immunoglobulin subclass, and titers of IgG1 correlate well with the level of NAbs22. As we set up cutoff points corresponding to 1:32 NAb titers, prevalence of IgG against AAV2 and AAV3B was lower than that reported in most previous studies where lower cutoff values were used; however, it is difficult to compare the results from different laboratories using non-standardized assays with variable sensitivities24. We also showed that the prevalence of IgG and titers of NAbs against AAV.GT5 were lower than those against AAV3B. Still, there were a substantial number of people who had low NAb titers against AAV.GT5. However, the lower antibody titers against AAV.GT5 can be mitigated by strategies such as evasion of pre-existing NAbs, as demonstrated on AAV5 in a hemophilia study24, or treatment with protease that degrades circulating IgG43, to harness the advantages of AAV.GT5 over other AAVs for clinical applications. Furthermore, high transduction efficiency and low reactivity toward NAbs may allow administration of relatively low doses, which could have potential benefits in terms of reduced immunogenicity and also allow for the inclusion of more number of patients in need of gene therapy.In the ELISA method used in this study, the AAV vector particles immobilized on plates captured AAV-specific antibodies. Using whole vector particles as antigens without degradation, antibodies that are specific to each AAV, including NAbs, can be assessedIn summary, we bioengineered AAV3 for efficient transduction of the vector into primary human hepatocytes and to alleviate the reactivity with the pre-existing NAbs without affecting its preparation yields.17: (1) located on the surface loop of variable regions; (2) located in or around the epitope of ant-AAV2 antibodies; (3) conserved between AAV3A and AAV3B; (4) less likely to affect capsid formation or genome incorporation; (5) expected to enhance transduction of human hepatocytes33. AAV vector plasmids contained an expression cassette consisting of a human cytomegalovirus immediate-early (CMV) promoter followed by cDNA encoding GFP or luciferase and a simian virus 40 polyadenylation signal sequence. The expression cassette was located between the inverted terminal repeats of the AAV3A genome. The AAV.GT5 vp cDNA was synthesized by introducing three amino acid substitutions  in AAV3B vp. For comparison, a single amino acid mutant AAV.M1 (S587A), and double amino acid mutants AAV.M2 (S472A and S587A) vp cDNAs were synthesized. Recombinant AAV vectors were produced by transient transfection of human embryonic kidney (HEK293) cells, as previously described44. The cells were maintained in Dulbecco’s Modified Eagle’s Medium and Harn’s F-12 Nutrient Mixture  supplemented with 10% heat-inactivated fetal bovine serum , 1% penicillin and streptomycin . The cells were transfected with the vector plasmid, the AAV3B rep and either AAV2 (Gene Bank Accession NC_001401.2), AAV3B (AF028705.1), AAV.GT5, AAV8 (NC_006261), AAV-Spark1003, or AAVhu37 (AY530600.1) vp expression plasmids, and the adenoviral helper plasmid pHelper (Agilent Technologies). The recombinant viruses were purified by isolation from two sequential continuous CsCl gradients. Viral titers were determined by qPCR. Empty-to-full particle ratios were determined by direct counting of the electron micrographs . Empty particles were distinguished based on the electron-dense centers following negative staining with uranyl acetate.To design AAV3 mutants based on the findings of previous AAV studies, we chose amino acid residues based on the following criteria18. HepG2 and Huh7 cells were maintained in DMEM low glucose (Thermo Fisher Scientific) with 10% FBS and 1% PenStrep. PXB cells were cultured in dHCGM  consisting of DMEM with 10% FBS, 20 mM HEPES, 44 mM NaHCO3, 1% PenStrep, 15 μg/mL l-proline, 0.25 μg/mL insulin, 50 nM dexamethasone, 5 ng/mL epidermal growth factor, 0.1 mM l-ascorbic acid, and 2% dimethyl sulfoxide.Human hepatocellular carcinoma HepG2 and Huh7 cells were purchased from the Japanese Collection of Research Bioresources Cell Bank . Primary human hepatocytes derived from chimeric mice with human liver (PXB mice) were obtained from PhoenixBio Co., Ltd. 4 cells per well in a 96-well optical bottom plates (Thermo Fisher Scientific). Twenty-four hours after seeding, HepG2 or Huh7 cells were infected with 5 × 108 vg/well of GFP expressing AAV2, AAV3B, AAV.GT5, and AAV8. PXB cells were plated at a density of 7 × 104 cells per well in a 96-well plate. Six days after seeding, PXB cells were infected with 5 × 108 vg/well of GFP expressing AAV2, AAV3B, AAV.GT5, and AAV8. For comparison of AAV.GT5, AAV-Spark100, and AAVhu37, 2 × 109 vg/well for HepG2 cells and 3.5 × 109 vg/well for PXB cells were infected. Seven to nine days after infection, GFP expression was measured using a plate reader .HepG2 or Huh7 cells were plated at a density of 5 × 10wild/+/SCID)18. Human hepatocytes were transplanted via the spleens. Only mice with serum human albumin levels were > 7.0 mg/mL (corresponding to > 70% humanized) were used for transduction experiments. AAV3B or AAV.GT5 vectors expressing the GFP transgene were diluted in 200 mL of phosphate-buffered saline (PBS) and injected into the 20-week-old male PXB mice through the tail vein at 1.0 × 1011 vg/mouse (n = 3 for each cohort).Mouse studies were approved by the Animal Care and Use Committee at Jichi Medical University (17203-02). All the methods were carried out in compliance with the relevant guidelines and regulations including the ARRIVE guidelines. PXB mice contained a transgene containing an albumin promoter/enhancer and urokinase-type plasminogen activator cDNA in a severe combined immunodeficient background , or human-specific CK8/18  in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. They were then incubated with Alexa Fluor 488 goat anti-chicken IgY  and Alexa Fluor 594 goat anti-mouse IgG  for 1 h at 24 °C. Immunoreactivity was assessed and viewed under a microscope  and using a confocal laser scanning microscope . The numbers of GFP and CK8/18 double-immunoreactive cells were counted in liver sections. For sections obtained from each of the three blocks, six fields of view at 72 × were counted. A total of 1254–1702 CK8/18-immunoreactive cells in three sections were sampled per animal for cell counting.6 vector genome (vg)/mL. Five microliters of diluted serum were added to 5 μL diluted AAV vectors, and the mixture was incubated for 1 h at 37 °C. The mixture was then added to 96-well plates containing 2 × 104 HEK293 cells. One day after transduction, luciferase expression was measured with a luciferase assay kit . The NAb titer was determined as the serum dilution at which 50% of AAV reporter gene expression is permitted, compared to no serum controls. For the comparison of inhibitory effects of NAbs against AAV.GT5, AAV.M1 (S587A), AAV.M2 (S472A and S587A), and AAV3B on transduction efficiency, GFP expression vectors were incubated with the serum containing NAbs against AAV2 (titer 1:32). Serum samples were surplus of blood donors for the Japan Red Cross Society (Tokyo). The samples were donated by an application for the use of blood donated in Japan based on the “Guidelines on the use of donated blood in R&D, etc.” Informed consent was obtained from all participants. All experiments were performed in accordance with these guidelines and approved by the institutional ethical committee of KAINOS Laboratories, Inc. (study number 2019-1). Details of the gender and age of the blood donors were not disclosed, however, they were healthy adults over 20 years of age and included both sexes. For the NAbs assay, heat-inactivated sera were continuously diluted two-fold with FBS. AAV2, AAV3B, and AAV.GT5 vectors that express luciferase under the CMV promoter were diluted with buffer containing 50 mmol/L HEPES and 150 mmol/L NaCl (pH 7.4) up to 1.6 × 1041. Ninety-six-well microtiter plates (Thermo Fisher Scientific) were coated with either 70 ng of AAV2, 500 ng of AAV3B, or 70 ng of AAV.GT5 vector particles per well. After blocking with 2% bovine serum albumin (BSA) in PBS, the plates were washed with 2% sucrose. Serum samples diluted at 1:1,000 with PBS/1% BSA were added to each well (100 μL/well). The plates were incubated for 1 h at 25 °C and washed three times with PBS/0.05% Tween 20. A horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG) was prepared using N-sulfosuccinimide, sodium salt  as a maleimide compound. A solution containing 0.1 μg/mL HRP-IgG was added to each well at a final concentration of 75 μg/mL for AAV2, 70 μg/mL for AAV3B, or 25 μg/mL for AAV.GT5. The plates were incubated for 1 h at 25 °C and washed three times with washing buffer. The color was developed by adding 100 μL 3,3′,5,5′-tetramethylbenzidine/urea hydrogen peroxide  and incubating the plates for 30 min at 20 °C. The color development was stopped by adding 1 mol/L sulfuric acid (100 μL/well), and the OD was measured at 450/640 nm.AAV-specific IgG were detected using an ELISA as previously describedTo examine the antibody avidity in five serum samples containing NAbs (titer > 1:32) against AAV2, AAV3B and AAV.GT5, guanidine thiocyanate or urea was added to each well at final concentrations of 0.9 M and 5.0 M, respectively before HRP-IgG was added to the ELISA plates.R software. Analysis of variance (ANOVA) was performed to determine differences for comparison among multiple experimental conditions. The Bonferroni post hoc test was used when comparing the conditions. Results are presented as mean ± SEM. Statistical significance was set at P < 0.05.Statistical analysis was performed using Supplementary Information.


Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs)  in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants  displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress , osmotic stress , cell wall-targeting agents , and fungicide . ΔGcn5, ΔSas3, and ΔHos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in ΔGcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, ΔRTT109, ΔGcn5, and ΔHos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in ΔGcn5 and ΔHos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) ( The fundamental structural subunit of the chromatin is the nucleosome, which consists of approximately 200 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3, and H4 . The revS. cerevisiae, the transcription of FLO1 was significantly reduced in the absence of both Sas3 and Ada2  complexes, such as ADA, SAGA, or SALSA . Gcn5 acand Ada2 . Rpd3 isand Ada2 . In yeasand Ada2 . Hos2 anand Ada2 . In yeasand Ada2 . Hos2 acand Ada2 . The yeaand Ada2 . The besand Ada2 .Aspergillus nidulans showed that GcnE (ortholog of Gcn5) controls asexual reproduction, and the biosynthesis of sterigmatocystin, penicillin and terrequinone A , F. graminearum (Elp3), and so on  Keissler used in the mutagenesis experiments was isolated from an infected citrus (Citrus suavissima Hort. Ex Tanaka) in Zhejiang, China .Citrus reticulata Blanco) leaves inoculated by placing a 5-mm dia. agar plug covered with fungal mycelium on each spot, and those inoculated leaves were kept in a plastic box at 26°C for 2–4 days for lesion development.Fungal virulence was assessed on detached Dancy , 1 M sorbitol, 1 M KCl, 1 M NaCl, 250 mM CaCl2, 1 mM CuSO4, 0.01% sodium dodecyl sulfate (SDS), 0.2 mg/ml congo red (CR), 10 mg/L chlorothalonil, 2 mg/L Mancozeb, 3 mg/L Boscalid, 5 mM hydroxyurea (HU), 0.1% methyl methanesulfonate (MMS), or 5 μM camptothecin (CPT). To examine the carbon utilization ability, mutants and wild-type were inoculated on MM medium using 20 g/L glucose, 10 g/L sucrose, 10 g/L starch, or 10 g/L lactose as sole carbon source. All tests were repeated at least twice with three replicates of each treatment.All the phenotypes of mutants and wild-type were evaluated. To examine the vegetative growth under multiple stresses, mutants and wild-type were inoculated on PDA containing 10 mM HGcn5 and ΔSas3 each with two replicates were cultured in PDB  and were harvested after 36 h. The samples were ground and homogenized in 1 ml lysis buffer composed of 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 μl protease inhibitor cocktail , 2 mM PMSF , and 50 mM Tris-HCl, pH 7.5. The homogenates were centrifuged at 14,000 rpm, 20 min, 4°C. The Supernatants were collected as total protein. Antibody used in this study is H3K4ac .Wild-type, ΔGcn5 and ΔHos2 each with two replicates were cultured in PDB  and were harvested after 36 h. RNA was extracted using Axygen RNA purification kit . The libraries were produced using an IlluminaTruSeq RNA Sample Preparation Kit, and they were sequenced on an Illumina HIseq 2500 platform, generating 150 bp paired-end reads. Trimmomatic Ver 0.36 (http://smart.embl-heidelberg.de/. Carbohydrate-active enzymes (CAZymes) were predicted using dbCAN meta server.1 The transcriptomes raw data  have been deposited in NCBI’s Sequence Read Archive.Wild-type, ΔVer 0.36  was usedVer 0.36  was usedVer 0.36 . DiffereVer 0.36  based onVer 0.36 . anti-SMVer 0.36  was usedAlternaria alternata reference genome with Bowtie2  was carried out on a 7300 Real Time PCR system  to validate the transcriptome data. RNA was extracted with an Axygen RNA purification kit  and the cDNA was synthesized from RNA using a PrimeScript RT regent kit . Actin-coding gene (KP341672) was used as an internal control and the resulting data were normalized using the comparative Cτ method as described previously .P < 0.05).The statistical significance of treatments was determined by analysis of variance and means separated by Duncan’s test , Elp3 (ΔElp3), RTT109 (ΔRTT109), Nat3 (ΔNat3), Sas3 (ΔSas3), Sas2 (ΔSas2), Hos2 (ΔHos2), Rpd3 (ΔRpd3), Hos3 (ΔHos3), Hda1 (ΔHda1), Hst2 (ΔHst2), and Sir2 (ΔSir2) were generated by the homologous recombination method. The mutants were verified by PCR analysis , indicating that most HATs play a vital role on the growth of A. alternata  than the wild-type   . Only ΔSed by Z7 . QuantitGcn5 on all tested sole carbon source media for 4 days was 100%. At the same time, the growth inhibition ratio of ΔSas3 on the MM medium supplemented with glucose, sucrose, lactose, and starch was increased by 28, 41, 39, and 41%, compared to the wild type. In contrast, ΔSas2, ΔElp3, and ΔRTT109 displayed wild-type sensitivity to all tested sole carbon source media. Most HDACs disruption mutants, including ΔSir2, ΔHos3, ΔHda1, and ΔHst2, exhibited wild-type sensitivity to all test medium. Only ΔHos2 showed slightly increased sensitivity on carbon sources of glucose and starch.To examine whether HATs or HDACs were required for the utilization of carbon sources, mycelial radical growth assays were performed on MM medium supplemented with sucrose, starch, glucose, or lactose as sole carbon sources . In HATsGcn5 on sole carbon source medium was not caused by its slow vegetative growth, the incubation time was extended to 10 days. Growth examination indicated that the growth inhibition ratio of ΔGcn5 on the medium with lactose as sole carbon source was still 100%, and only a few hyphae of ΔGcn5 appeared on carbon sources of sucrose, starch, or glucose (the growth inhibition ratio is close to 100%), confirming that the Gcn5 was required for the utilization of carbon sources (To prove further that the severe growth defects of Δ sources .Elp3 and ΔGcn5 were more sensitive to H2O2 and CHP (cumyl-H2O2), while ΔSas3 and ΔRTT109 just showed increased sensitivity to H2O2, compared with wild-type. In addition, ΔSas3 showed elevated sensitivity to cell wall-targeting agents such as Congo red (CR) and sodium dodecyl sulfonate (SDS). At the same time, ΔElp3, ΔGcn5, and ΔRTT109 just displayed increased sensitivity to CR. ΔSas3, ΔGcn5, and ΔRTT109 displayed increased sensitivity to NaCl, KCl, and CuSO4. Only ΔRTT109 displayed increased sensitivity to CaCl2. However, ΔSas3, ΔElp3, and ΔGcn5 displayed enhanced resistance to CaCl2 compared to wild-type. Both ΔSas3 and ΔElp3 also showed increased tolerance to V8 medium compared to wild-type. Only ΔSas2 showed wild-type resistance to all test chemicals , Amino acid transport and metabolism (E), Lipid transport and metabolism (I), and Inorganic ion transport and metabolism (P). There are also many DEGs in ΔGcn5 or ΔHos2 enriched in Transcription (K) and Replication, recombination and repair (L)  analysis of Δpair (L) , 9. KEGGynthesis , 9. AlthGcn5 and Hos2 are involved in regulating the transcription of many genes involved in carbon (sugar) source utilization. Analysis of DEGs in Glycolysis/Gluconeogensis (ko00010), Fructose and Mannose Metabolism (ko00051), Galactose Metabolism (ko00052), Starch and Sucrose metabolism (ko00500), Pentose Phosphate Pathway (ko00030), Citrate cycle (ko00020), and Pentose and Glucuronate interconversions (ko00040) in ΔGcn5 and ΔHos2 revealed that the number of down-regulated genes  was significantly greater than the up-regulated genes   . In yeasos2, 15) . In A. aGcn5 and Hos2 affect the expression of ROS detoxification genes. In view of the increased sensitivity of ΔGcn5 and ΔHos2 to oxidative stress, we analyzed the transcription level of 96 ROS detoxification genes, including 7 catalases, 7 SOD superoxide dismutases, 42 glutathione system-related genes, 20 thioredoxin system-related genes, and 20 peroxidases , Nucleotide excision repair (NER), Mismatch repair (MMR), Homologous recombination (HR), and Non-homologous end-joining (NHEJ) play important roles in the repair of damaged DNA in organisms. Transcriptome analysis revealed that BER, NER, MMR, or HR-related genes including UDG , Polδ , and TFIIH  were down-regulated in ΔGcn5  polymerase, AALT_g9674], and NER related gene XPA  were down-regulated  in ΔHos2 were up-regulated ” were up-regulated, including protein glycosyltransferase (AALT_g5706), Mannosyl-oligosaccharide glucosidase (AALT_g11241), Mannosyl-oligosaccharide alpha-1,2-mannosidase (AALT_g2989), HSP20 (AALT_g9536), UBC5 (AALT_g6888), UBC7 (AALT_g619), and RBX1 (AALT_g4961), which are involved in protein processing and degradation. Furthermore, eight up-regulated genes were enriched in “Ubiquitin mediated proteolysis (ko4120)” and “Regulation of Autophagy (ko04140)” , “Ubiquitin mediated proteolysis” (AALT_g6960), and “Regulation of Autophagy” were up-regulated (AALT_g11308) (o04140)” . Only 4 _g11308) .Gcn5 or Hos2 also affected the transcription of genes involved in Carbohydrate-active enzymes (CAZymes), secondary metabolite cluster, CYP450, and conidiation , located in the ACT toxin gene cluster and required for pathogenicity but plays no role for ACT toxin synthesis (Gcn5 (FC = −25) and ΔHos2 (FC = −6).In addition, deletion of idiation . The traynthesis , was sigGcn5, Hos2, and RTT109 are required for DNA damage repair in B. bassiana and S. cerevisiae , hydroxyurea (HU) and methyl methanesulfonate (MMS) in A. alternata. Compared with wild-type, growth of ΔRTT109 was significantly suppressed by 40 and 89% on PDA containing 5 μM CPT or 0.1% MMS, respectively  but displayed wild-type resistance to 5 μM CPT  irradiated ΔGcn5 and wild-type. In the Gcn5 coding region, no reads were detected in ΔGcn5, but many reads were found in wild-type, which proves once again that the knock-out experiment of Gcn5 was successful  in the ΔGcn5 compared to Z7. No reduction of the signal for H3K4ac was observed in ΔSas3.To test whether histone acetylation levels were altered in ΔA. alternata. To systematically elucidate the functions of HATs and HDACs in A. alternata, we created 6 HATs and 6 HDACs deletion mutants. Our studies showed that HATs and HDACs were widely involved in biological processes of mycelial growth and development, conidial production, multiple stresses resistance, carbon source utilization, DNA damage repair, and pathogenicity in A. alternata.Histone acetylation, which is important for multiple cellular processes, occurs in different organisms ranging from fungi to mammals, and the acetylation level manifested by HATs and HDACs dynamically changes over time during development and differentiation . In planGcn5, ΔHos2, ΔRpd3, ΔNat3, ΔSas3, and ΔRTT109 produce no or fewer conidia. In addition, ΔGcn5, ΔRpd3, and ΔNat2 produces more hyphal branches than wild-type. In some filamentous fungi like N. crassa and A. nidulans, the central development pathway relies upon the key activator genes BrlA, AbaA, and WetA  are required for the activation of BrlA and the initiation of conidiation  , F. graminearum , and S. cerevisiae (Nat3)  . Other flternata . Whethere (Nat3) .Gcn5, ΔSas3, ΔRTT109, and ΔHos2 are required for cellular resistance to osmotic stress. In yeast, SAGA (Spt-Ada-Gcn5) is required for the transcription of Hog1-mediated genes under severe osmostress  and inhibit the utilization of other carbon sources  were not significantly differentially expressed in ΔGcn5 and ΔHos2 compared to wild-type. However, the transcription of SUC2 (AALT_g2401), which is regulated by Snf1 and is essential for sucrose utilization in S. cerevisiae  metabolism, which is one of the most important biological processes in organisms, is involved in growth, development, and multiple stress resistance . This st sources . In B. blization , d. In a sources . Sucrose sources . In Fusanhibited . SNF1 corevisiae , was dowcription . In addilternata . TherefoGcn5, Sas3, Elp3, RTT109, or Hos2 in A. alternata were defective in ROS detoxification at varying degrees, indicating all of these five genes are involved in resistance to oxidative stress. RTT109, Gcn5, and Hos2 in B. bassiana, Elp3 and Sas3 in F. graminearum also play important roles in the resistance to oxidative stress  is essential for folding and trafficking of proteins that enter the secretory pathway, while the disorder of redox homeostasis of ER could result in protein misfolding  or HDACs (Hos2) displayed increased sensitivity to DNA-damaging stresses, indicating that all of these genes are involved in DNA damage repair  can be found in the article/HM, FG, and HL contributed to the conception of the study. HM, LL, YG, XYZ, YNC, XKZ, and YZC performed the experiments. HM, YG, and CJ performed the data analysis. HM wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Magnaporthe oryzae avoids T6P accumulation and the genetic and physiological adjustments it undergoes to self-adjust the metabolite level when it is unavoidably accumulated. We found that T6P accumulation leads to defects in fugal development and pathogenicity. The accumulated T6P impairs cell wall assembly by disrupting actin organization. The disorganization of actin impairs the distribution of chitin synthases, thereby disrupting cell wall polymer distribution. Additionally, accumulation of T6P compromise energy metabolism. M. oryzae was able to overcome the effects of T6P accumulation by self-mutation of its MoTPS3 gene at two different mutation sites. We further show that mutation of MoTPS3 suppresses MoTps1 activity to reduce the intracellular level of T6P and partially restore ΔMotps2 defects. Overall, our results provide insights into the cytotoxicity effects of T6P accumulation and uncover a spontaneous mutation strategy to rebalance accumulated T6P in M. oryzae.Trehalose biosynthesis pathway is a potential target for antifungal drug development, and trehalose 6-phosphate (T6P) accumulation is widely known to have toxic effects on cells. However, how organisms maintain a safe T6P level and cope with its cytotoxicity effects when accumulated have not been reported. Herein, we unveil the mechanism by which the rice blast fungus IMPORTANCEM. oryzae, the causative agent of the rice blast disease, threatens rice production worldwide. Our results revealed that T6P accumulation, caused by the disruption of MoTPS2, has toxic effects on fugal development and pathogenesis in M. oryzae. The accumulated T6P impairs the distribution of cell wall polymers via actin organization and therefore disrupts cell wall structure. M. oryzae uses a spontaneous mutation to restore T6P cytotoxicity. Seven spontaneous mutation sites were found, and a mutation in MoTPS3 was further identified. The spontaneous mutation in MoTPS3 can partially rescue ΔMotps2 defects by suppressing MoTps1 activity to alleviate T6P cytotoxicity. This study provides clear evidence for better understanding of T6P cytotoxicity and how the fungus protects itself from T6P’s toxic effects when it has accumulated to severely high levels. Trehalose is a nonreducing sugar that consists of two glucose units that are jointed together by an α-1,1-glycosidic bond. It is a structural component of most organisms, including plants, fungi, lichens, algae, bacteria, insects, and invertebrates, but not vertebrates . Therefo4–7–Magnaporthe oryzae is the causative agent of the rice blast disease , trehalose 6-phospahte phosphatase (MoTps2), and a regulatory subunit (MoTps3)  . A previn source . MoTps1 n source . The glun source . In addinfection .M. oryzae. Nevertheless, TPS2 orthologs are well conserved in other fungi, and several of them have been characterized in fungal pathogens. In Fusarium graminearum, loss of the FgTPS2 gene significantly affects the growth, sporulation, and virulence of the fungus, and the ΔFgtps2 mutant is defective in mycotoxin production  cycle metabolites, as well as amino acid biosynthesis. In addition, the TPS complex possesses a spontaneous “correction” way to modulate T6P homeostasis by spontaneous mutation of the regulatory subunit MoTps3. This spontaneous correction function further suppresses the MoTps1 activity to downregulate T6P, resulting in partial restoration of the ΔMotps2 mutant’s defects in growth, conidiation, and pathogenicity. This study presents new insights into the cytotoxicity of T6P accumulation and uncovers a spontaneous way of correcting of T6P accumulation cytotoxicity in M. oryzae.In the present study, we used genetic approaches to analyze the biological functions of MoTps2 in the rice blast fungus. Our results indicate that deletion of MoTPS2 (MGG_03441) is the only TPS2 homologue in M. oryzae, and it encodes a 1,020-amino-acid (aa) protein that shares 33.4% identity in amino acid sequences with Saccharomyces cerevisiae Tps2p  by replacing MoTPS2 with a hygromycin resistance cassette (HPH), using homologous recombination, and further confirmed the deletion by PCR analysis and Southern blot assay (see Motps2 mutant background. The wild type strain (70-15), mutant (ΔMotps2), and complemented strain (ΔMotps2-com) were then cultured on complete medium (CM), minimal medium (MM), and rice bran medium (RBM) for 7 days to compare their growth rates. The ΔMotps2 mutant showed significant reductions (P < 0.01) in growth rate on these media compared to the 70-15 and ΔMotps2-com strains  Amino acid alignment of the trehalose 6-phosphate phosphatase domain in different species. Download FIG S1, TIF file, 2.0 MB.Comparative analysis of Tps2 protein in fungi. (a) Phylogenetic tree and putative domain structures of Copyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00462-21.2FIG S2Motps2 mutants (a), ΔMotps1 mutants (b), and ΔMotps2ΔMotps1 mutants (c). Download FIG S2, TIF file, 1.2 MB.PCR analyses and southern blot assays of the ΔCopyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00462-21.3FIG S3P < 0.01. Download FIG S3, TIF file, 1.8 MB.MoTps2 is required for normal growth on different carbon sources. Photos were taken after 7 days. **, Copyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Motps2 mutant had a significant (P < 0.01) reduction in conidiation . The ΔMotps2 mutant was normal in germination, but only about 2.5% of its germ tubes formed appressoria at 24 hpi  at their hyphal tips when inoculated on hydrophobic surfaces  indicated that the cell wall of the ΔMotps2 mutant was obviously thicker than that of the wild type  results  Actin organization is essential for proper chitin deposition. Treatment with Lat A leads to accumulation of chitin at hyphal tips. Bar = 10 μm. Download FIG S5, TIF file, 2.9 MB.MoTps2 is important for cell wall homeostasis. (a) Loss of MoTps2 alters the expressions of melanin-, glucan-, and chitin-related genes. To monitor the expression of cell wall structure-related genes, the wild type and ΔCopyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CHS) are responsible for the production of chitin polymers, and CHS1, CHS3, and CHS4 genes have been shown to be involved in cell wall synthesis and maintenance of cell wall integrity in the filamentous fungus Neurospora crassa  fusion protein in both strains. An actin patch was found to accumulate at the Spitzenkörper and subapical collar in the wild type. In the Δhal apex . In yeashal apex , 43. In d growth . We there mutant . Consistblotting , which cMotps2 mutant was not very stable and eventually produces spontaneous suppressor strains with a higher growth rate than the mutant (see Motps2-m) was partially rescued in the defects of the ΔMotps2 mutant in growth, hyphal morphology, conidiophore formation, and pathogenicity. Moreover, TEM examination revealed that the ΔMotps2-m strain had a similar cell wall thickness to the wild type (We observed that the Δild type .10.1128/mSystems.00462-21.6FIG S6Motps2-m mutant. (a) Spontaneous suppressor strain of the ΔMotps2 mutant. The fast-growing sector is indicated by a red arrow. (b) Spontaneous mutations identified in two suppressor strains through whole-genome sequencing. Download FIG S6, TIF file, 2.5 MB.Identification of the spontaneous mutation sites in the ΔCopyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Motps2-m spontaneous suppressor strains could be restored in actin organization and distribution of chitin synthase-related proteins, thereby rescuing ΔMotps2 defects. To validate this hypothesis, we also expressed the Lifeactin-RFP, MoChs1-GFP, MoChs3-GFP, and MoSpa2-GFP fusion proteins in ΔMotps2-m strains. Confocal microscopic examination showed that the localizations of actin and chitin synthase-related proteins in the ΔMotps2-m strain are similar to those in the wild type. In addition, the localization of MoSpa2 is also rescued in the ΔMotps2-m mutant (We speculated that the Δm mutant .MoTPS3 genes (MGG_14118), the regulatory subunit of the TPS complex. The spontaneous mutant strains have 2 base deletions at C1930 and G1931 of the GT20-TPS (glycosyltransferase family 20) domain and 1 base insertion (base A) between A3139 and T3140 of the OtsB  domain of the MoTPS3 open reading frame, which could have led to the loss of MoTps3 activity , MGG_05689 (base transition), MGG_07789 (base transition), MGG_08555 (base transversion), MGG_01956 (base transition), MGG_03973 (base transversion), and MGG_14118 (base deletion and base insertion) . We notiactivity . To confn plants .M. oryzae, MoTps3 is the only regulatory subunit of the TPS complex, and previous studies have indicated that MoTps3 can regulate MoTps1 activity  suppresses MoTps1 activity and therefore partially restores ΔMotps2 defects in growth, conidiogenesis, cell wall structure, and pathogenicity.To further characterize the suppression mechanism of MoTps3, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were carried to identify the various interactions among the TPS complex subunits. As shown in t assays b and c. position . Moreoveild type . Since aild type , we concMoTPS1 or MoTPS3 in the ΔMotps2 mutant can partially rescue its defects, we then assayed T6P and trehalose production. The key metabolites in the TPS complex pathway are trehalose and trehalose 6-phosphate. If the low level of the final product  is the major reason that led to the phenotypic defects of the ΔMotps2 mutant, then the ΔMotps1 mutant should have more severe defects than the ΔMotps2 strain, since the ΔMotps1 strain has less trehalose than the ΔMotps2 strain  (Escherichia coli (Motps2 mutant (Motps2 mutant (Motps2 defects (Motps2 mutant (Motps2-m and ΔMotps2ΔMotps3 mutants (Motps2 was mainly due to the accumulation of T6P in the mutant.To further understand why deletion of 2 strain . However defects . In addiosphate) , from Eshia coli  and gene2 mutant . Microsc2 mutant . Further defects . In addi2 mutant . Coupled mutants , we conc10.1128/mSystems.00462-21.7FIG S7Motps2 growth defect. (b) Exogenous trehalose 6-phosphate has no effect on vegetative growth, conidial germination, and appressorial formation. Download FIG S7, TIF file, 1.5 MB.Effect of exogenous trehalose or trehalose 6-phosphate on fungal development. (a) Exogenous trehalose failed to rescue the ΔCopyright © 2021 Chen et al.2021Chen et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Motps2 mutant.To gain more insights into the effects of trehalose 6-phosphate accumulation in the fungus, the wild type was cultured on CM plates with or without trehalose 6-phosphate. The results indicate that exogenous T6P has no obvious effect on the vegetative growth of the fungus , most liM. oryzae  in vegetative hyphae were significantly lower than those of the wild type  and the TCA cycle  compared to the wild type. To better understand the relationship between MoTPS2 deletion and the metabolites’ abundance, we checked the primary metabolite levels in the ΔMotps2-m, ΔMotps2ΔMotps1, and ΔMotps2-TreC strains. Interestingly, most of the metabolite levels were restored when the intracellular T6P level was reduced    and f, sM. oryzae, the causal agent of the rice blast disease, and uncovered a spontaneous correction way of the TPS complex to modulate T6P homeostasis  enzymes. The chitin synthases are divided into seven families in most filamentous fungi, and most of them have been shown to localize at the hyphal apex , which in turn suppresses MoTps1 activity to reduce the level of T6P. It will be interesting to understand the underlying mechanism of how MoTps3 senses the accumulation of intracellular T6P and induces self-mutation to compromise the defects caused by T6P accumulation.The most widely distributed trehalose biosynthesis pathway contains two key enzymes—trehalose 6-phosphate synthase (Tps1) and trehalose 6-phosphate phosphatase (Tps2)—in a complex called the TPS complex , 67. In nditions . This su2 mutant  and e. F defects  and d. S defects , 70, we MoTPS2, has toxic effects on the cell and results in growth, conidiation, and pathogenicity defects. Nevertheless, M. oryzae can alleviate these defects through induction of spontaneous mutation of the MoTPS3 gene. Our results suggest that the spontaneous mutation of MoTPS3 further suppresses MoTps1 activity. Under this condition, the biological functions of the TPS complex subunits are almost suppressed. However, the spontaneous mutation strain has no significant difference in growth compared to the wild type and can also produce conidia and pathogenicity lesions  from the various strains were placed on hydrophobic coverslips for 2, 4, 6, and 24 h and finally observed under a microscope. For development of appressorium-like structures, mycelial balls were harvested from 2-day-old CM agar cultures and then placed on hydrophobic coverslips, paraffin wax, and barley and rice leaves for 72 h, respectively. For the penetration assay, hyphal blocks of the indicated strains were placed on 7-day-old barley leaves, incubated at 28°C for 36 h, and then observed under a confocal microscope. For the plant infection assay, mycelial plugs were placed on intact or wounded barley leaves and incubated in a humid chamber at 28°C for 5 days.To induce the formation of appressoria, 20-μl conidial suspensions  was amplified and cloned into a pKNT-RP27-GFP vector. The resulting fusion vectors were further verified by sequence analysis .For construction of MoChs1-GFP and MoChs3-GFP vectors, the various genes encoding the er pairs  and thenFor observation of hyphal morphology, the various strains were cultured on CM agar plates for 7 days, after which hyphal blocks were placed upside down on coverslips and used for further confocal microscopic examinations. For staining of the chitin component of the cell wall, fluorescent brightener 28 was used at a final concentration of 10 μg/ml to stain the fungal hyphae in preparation for confocal microscopy. To examine the effect of actin organization on the localization of chitin synthases, the actin inhibitor latrunculin A (Lat A) (Sigma-Aldrich) was used at a final concentration of 10 μg/ml to treat the various strains. Microscopic examinations were performed using a Nikon A1 laser confocal microscope .Ex Taq II (TaKaRa) using specific primers (−ΔΔCT) method . The sequence reads were mapped onto the reference genome of strain 70-15 . The PrimeScript RT reagent kit (TaKaRa) was used for cDNA synthesis; the transcription levels of the genes were detected using TB Green Premix  primers . β-Tubul) method . For whoin 70-15 . SV  were added, and the mixture was incubated at 70°C for 20 min and finally centrifuged. The supernatants were collected, and 500 μl ddH2O and 268 μl chloroform were added. The mixture was centrifuged at 12,000 rpm for 10 min, and the resulting supernatant was transferred into a 2-ml tube and dried in a freeze dryer. About 50 μl methoxyamine-pyridine mixture was added into the tube, and the mixture was kept at 30°C for 90 min. Afterwards, 80 μl BSTFA  plus 1% TMCS (trimethylchlorosilane) was added, and the mixture was incubated at 65°C for 60 min. The final samples were transferred to a GC-MS glass vial and then analyzed with a Gerstel autosampler, an Agilent 7890B gas chromatograph, and Pegasus HT time-of-flight mass spectrometer . Trehalose 6-phosphate dipotassium salt (Santa Cruz) and d-(+)-trehalose dehydrate (Sigma-Aldrich) were used as standards.For the measurement of trehalose and trehalose 6-phosphate, GC-MS analysis was performed as previously described with small modifications , 79. Thet test. Values are presented as means ± standard error (SE), where * indicates P < 0.05 and ** indicates P < 0.01.Statistical analyses were performed using GraphPad Prism 7 . Data from three biological replicates were analyzed using the unpaired two-tailed Student's


Its strength of association with cardiovascular outcomes in the general population remains uncertain. This study aims to assess the association of HbA1c with cardiovascular outcomes in the general population.Biomarkers may contribute to improved cardiovascular risk estimation. Glycated hemoglobin A1c was evaluated in conjunction with classical cardiovascular risk factors (CVRFs) for association with cardiovascular mortality, cardiovascular disease (CVD) incidence, and overall mortality in subjects without diabetes  and with diabetes (N = 3684).Data from six prospective population-based cohort studies across Europe comprising 36,180 participants were analyzed. HbA1c levels (log-rank-test: p < 0.001). Cox regression analysis revealed significant associations between HbA1c (in mmol/mol) in the total study population and the examined outcomes. Thus, a hazard ratio (HR) of 1.16  1.02–1.31, p = 0.02) for cardiovascular mortality, 1.13  for CVD incidence, and 1.09  for overall mortality was observed per 10 mmol/mol increase in HbA1c. The association with CVD incidence and overall mortality was also observed in study participants without diabetes with increased HbA1c levels  and HR 1.10; 95% CI 1.01–1.20, p = 0.02) respectively. HbA1c cut-off values of 39.9 mmol/mol (5.8%), 36.6 mmol/mol (5.5%), and 38.8 mmol/mol (5.7%) for cardiovascular mortality, CVD incidence, and overall mortality, showed also an increased risk.Kaplan–Meier curves showed higher event rates with increasing HbA1c is independently associated with cardiovascular mortality, overall mortality and cardiovascular disease in the general European population. A mostly monotonically increasing relationship was observed between HbA1c levels and outcomes. Elevated HbA1c levels were associated with cardiovascular disease incidence and overall mortality in participants without diabetes underlining the importance of HbA1c levels in the overall population.HbAThe online version contains supplementary material available at 10.1186/s12933-021-01413-4. Despiandidate . The pril for DM , 8.1c levels and the risk of cardiovascular outcomes or overall mortality have been reported [1c may be associated with cardiovascular outcomes in an apparently healthy population [1c levels in middle-aged individuals without a history of CVD and HbA1c levels in the nondiabetic range [1c levels and an increased risk of coronary artery disease [1c might be associated with an increased risk and could be of importance in individuals without a diagnosis of diabetes [While associations between HbAreported –14, onlypulation , 15–19.  disease , 21. In 1c levels in population-based cohorts across Europe. Furthermore, we analyzed the association of continuous HbA1c levels with cardiovascular mortality, CVD incidence, and overall mortality. In addition, the association between HbA1c levels and time-to-event was analyzed in subgroups with and without diabetes and according to age. Finally, cut-offs for the dichotomization of HbA1c were determined for each outcome.In the present study, we evaluated the distribution of HbA1c levels.The design and rationale of the Biomarker for Cardiovascular Risk Assessment across Europe (BiomarCaRE) project have been described previously . BrieflyThe present analysis included six cohort studies from four European countries , namely the Cooperative Health Research in the Region of Augsburg (KORA) Study, the Study of Health in Pomerania (SHIP), and the Epidemiologische Studie zu Chancen der Verhütung, Früherkennung und optimierten THerapie chronischer ERkrankungen in der älteren Bevölkerung (ESTHER) Study, all from Germany, the MONICA Brianza Study from Italy, the Northern Sweden MONICA Study from Sweden, and the Tromsø Study from Norway. Each cohort is based on a well-defined population  were assigned to the DM group. Additional sub-classification into type 1 or type 2 DM was not possible with this dataset. Treatment of DM was available as (a) medication with insulin, (b) oral medication, but no insulin and (c) diet only.For each cohort, the following harmonized variables were available at baseline: duration of time with diagnosed diabetes in years, age, sex, smoking status, body-mass-index (BMI), systolic blood pressure, diastolic blood pressure, total cholesterol, high-density lipoprotein (HDL) cholesterol, HbASmoking status was determined based on self-reports. BMI, systolic blood pressure, diastolic blood pressure, total cholesterol, and HDL cholesterol parameters were measured (high blood pressure was defined either as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg) and it was recorded whether the patient was on antihypertensive medication. Prevalent CVD, like previous myocardial infarction or stroke, was assessed using the documented or self-reported history of myocardial infarction or stroke, including angina pectoris when the data did not permit its distinction from myocardial infarction. In MORGAM, prevalent heart failure was assessed with the item documented or self-reported history of heart failure.The following outcome measures were defined: (I) cardiovascular mortality, (II) CVD incidence, and (III) overall mortality, defined as mortality due to any cause during follow-up. Follow-up commenced at the baseline examination date .Cardiovascular mortality included death due to coronary heart disease or stroke. Cardiovascular disease, as an endpoint, was defined as the first fatal or non-fatal coronary event or possible ischemic stroke. Coronary events included acute definite or possible myocardial infarction or coronary death, unstable angina pectoris, cardiac revascularization, and unclassifiable death . In the MONICA/KORA Augsburg study, cardiac revascularization was not followed-up. In the MONICA/KORA Augsburg and MONICA Brianza studies, unstable angina pectoris was not assessed as an outcome but primarily included in the category “possible myocardial infarction” of the WHO MONICA classification used in these studies.1c was measured using whole blood. All HbA1c measurements were performed upon study entry to avoid glycation of blood samples during storage (except the Northern Sweden MONICA cohort measuring HbA1c levels in samples that had been stored at − 80 °C). Locally measured HbA1c values were transferred directly to the MORGAM Data Center (except for the SHIP study). The assays either reported their results as percentages (%), following the National Glycohemoglobin Standardization Program (NGSP), or in units of mmol/mol if they had employed the International Federation of Clinical Chemistry (IFCC) consensus reference method. Data from cohorts which reported their values as percentages were converted to mmol/mol using the standard formula: IFCC = 10.93 * NGSP − 23.50.HbA1c tertiles. For the age-adjusted Kaplan–Meier survival analyses the three HbA1c tertiles were < 34.4 mmol/mol (5.3%), 34.4 mmol/mol (5.3%) − 38.8 mmol/mol (5.7%); and > 38.8 mmol/mol (5.7%). To adjust the survival curves for the age distribution in the data, the following procedures were applied: (a) age was categorized using cut-offs 35, 40, 45, 50, 55, 60, 65, and 70 years; (b) an individual belonging to a particular HbA1c tertile and to age category j was assigned a weight equal to (nj/N)/(nij/N), where N is the total sample size, nj is the number of individuals in age category j, and nij is the number of individuals in age category j belonging to the HbA1c tertile; (c) these weights were then applied to the observations for estimating the Kaplan–Meier curves. A log-rank test was used to compare the unadjusted survival curves. The adjusted survival curves we compared using a robust score test obtained from a weighted Cox model with HbA1c categorized using tertiles as the only predictor. Follow-up time quartiles were estimated by the Kaplan–Meier potential follow-up estimator [Unadjusted and age-adjusted Kaplan–Meier survival curves for cardiovascular mortality, CVD incidence, and overall mortality were computed based on HbAstimator .1c (in mmol/mol) was applied untransformed and used as continuous variable. The Cox models for the three endpoints were adjusted for age , sex and cohort (strata), and CVRFs, smoking status (daily smoker yes/no), BMI, systolic blood pressure, DM (yes/no), total cholesterol to HDL cholesterol ratio and treatment of DM. Two separate extensions of the models were considered. In one extension, baseline age groups < 55, 55–64, ≥ 65 years at baseline and their interaction with continuous untransformed HbA1c was added to the models. In the second extension, an interaction between continuous untransformed HbA1c and the group indicator for diabetes was added. For these models results the HbA1c hazard ratios are presented in forest plots and tables. In the former HbA1c hazard ratios are presented per 10 mmol/mol increase and in the later per 1 mmol/mol increase  [A two-sided P-value of < 0.05 was considered statistically significant. Adjustment for multiple testing was not performed due to the exploratory nature of the analyses . All staect.org) .1c measurements for the entire cohort and for individuals with and without DM are shown in Table The baseline characteristics for individuals with available HbA1c measurements and information regarding diabetes status were available. Men and women were represented almost equally . The median age was 57.4 years, the median BMI 26.4 kg/m2, and the median systolic blood pressure 133.5 mmHg. At baseline, approximately 28% of the study cohort were daily smokers, 47.5% had high blood pressure or were taking antihypertensive medication, and 10.2% had a diagnosis of diabetes.For 36,180 subjects, HbA1c levels in the entire cohort and each cohort study are shown in Additional file 1c was 36.6 mmol/mol (5.5%). The 25th and 75th percentiles were 32.2 mmol/mol (5.1%) and 39.9 mmol/mol (5.8%), respectively.The distribution of HbAThe maximum follow-up time was 21.9 years. During the median follow-up time of 9.9 years, 1392 cases of cardiovascular death, 2711 cases of CVD, and 4,601 deaths due to any cause were observed. Further information on the median follow-up for each cohort is provided in Additional file 1c tertiles < 34.4 mmol/mol (5.3%), 34.4 mmol/mol (5.3%) − 38.8 mmol/mol (5.7%); and > 38.8 mmol/mol (5.7%) the probability of all investigated outcomes increased with increasing HbA1c levels  shows associations with cardiovascular mortality, CVD incidence, and overall mortality, with respective HR of 1.16 : 1.02–1.31, p = 0.02) for cardiovascular mortality, 1.13  for CVD incidence, and 1.09  for overall mortality per 10 mmol/mol increase in HbAIn individuals with DM, we observed a HR of 1.19  for cardiovascular mortality, 1.17  for CVD incidence, and 1.08  for overall mortality. In participants without DM, the respective HRs for cardiovascular mortality, CVD incidence, and overall mortality were 1.14 , 1.12 , and 1.10   but diminished with increasing age. In this context, the results for the cohort with subjects older ≥ 55 years and < 65 years were  and not present in individuals older 65 years  for cardiovascular disease  .Modelling the association of HbA2%) Fig. . The hyp1c cut-off values for cardiovascular mortality, CVD incidence, and overall mortality were calculated, yielding 39.9 mmol/mol (5.8%), 36.6 mmol/mol (5.5%), and 38.8 mmol/mol (5.7%), respectively. These cut-offs indicate an increased risk for the outcome. This is shown by the age-adjusted Kaplan–Meier curves for the three outcomes , especially since all our calculated cut-offs for subject differentiation were below the reported threshold to diagnose diabetes. The cut-off of > 38.8 mmol/mol (5.7%) is also in line with the threshold proposed by the American Diabetes Association to define prediabetes in individuals without a diagnosis of diabetes [1c levels above the cut-off value of 36.6 mmol/mol (5.5%) were associated with cardiovascular diseases. This underlines possible implications for levels below the cut-off value of HbA1c used to diagnose prediabetes (i.e. 5.7%).In this context, the current results with cut-off analysis adjusted for age provided thresholds of HbAociation  concerniidelines  on adultidelines , 30, 31.d before . Further1c levels for the association with CVD [1c levels in the clinical practice, additional data pointing out the usage of HbA1c levels for risk stratification in patients with CVD or DM with an insufficient glucose control in patients from Asia should be noted [A recent study in a smaller study population from Spain could also show the additional benefit of including HbAbe noted , 33, 34.1c levels and cardiovascular outcome [The recent study by Welsh and colleagues using data from the UK only with a median follow-up of 8.9 years also showed an association between HbA outcome . The aut outcome . However outcome .1c and cardiovascular disease incidence was strongest in individuals aged under 55 years. This could imply that young individuals with elevated HbA1c levels may carry an additional risk for developing relevant cardiovascular disease. A possible causal link between HbA1c levels and an increased risk of coronary artery disease was proposed in Mendelian randomization studies [An important finding was that the association between HbA studies , 21. Alt studies . Further1c with the risk of cardiovascular mortality, cardiovascular disease incidence, and overall mortality was mostly monotonic, considering the broad range of HbA1c levels encountered in such large population-based studies [1c levels in participants without diabetes. In addition, the calculated cut-offs showing an elevated risk for overall mortality in individuals with > 38.8 mmol/mol (5.7%) highlights the importance of elevated HbA1c levels and might indicate an increased risk for cardiovascular outcomes and overall mortality. In the context of the current findings, previous studies reported a poor outcome for patients with coronary artery disease, atrial fibrillation or individuals with DM and either cardiovascular risk factors or coronary artery disease [1c with multiple drugs [In this large European general population sample the association of HbA studies , 28, 37. studies , and the disease –41. Howele drugs , 43.1c levels might be of benefit for identifying individuals with a high risk of developing DM and/or CVD, especially in the presence of risk factors such as obesity, dyslipidemia and/or arterial hypertension. In this context the American Diabetes Association recommends to measure HbA1c levels in asymptomatic young adults [It has to be pointed out that measurement of HbAg adults .1c levels into assessment of risk prediction regarding the defined outcomes did not show an improvement as reflected by C-indices. This is inline with previous reported results [Inclusion of HbA results , 28.The present study has several strengths and limitations. An important strength is the considerable size of the dataset, with harmonized data from well-defined European population-based cohort studies with a long follow-up time.1c levels above 48 mmol/mol/6.5% that had not been diagnosed with diabetes. As the omission of such a sizable group may introduce considerable errors into our findings, we decided to classify individuals with HbA1c levels above 48 mmol/mol/6.5% and without the diagnosis of diabetes as subjects with prevalent diabetes. An additional limitation is the heterogeneity of data. Several cohort studies whose data we used commenced in the 1980s and 1990s, when treatment options and guidelines differed substantially from today’s. In population-based cohort studies with apparently healthy individuals, selection bias is a common problem.Despite the well-defined dataset, we identified 717 individuals with HbA1c which may affect our results [Haemoglobinopathies, different ethnicities, and certain disease states like bleeding, transfusion, or hemodialysis can interfere with the measurement of HbA results , 44.1c. These parameters were reported to have a prognostic impact and might be better for risk stratification than HbA1c [Due to the absence of additional measures of dysglycemia like 2-h post load glucose and fasting glucose we could not perform additional analyses to prove and to validate the prognostic impact of HbAan HbA1c , 46.1c from several European countries. HbA1c levels were positively associated with an increased risk for cardiovascular mortality, CVD, and overall mortality. There was a mostly monotonically increasing association between HbA1c levels and time-to-event regarding the defined endpoints cardiovascular mortality, cardiovascular disease incidence, and overall mortality, emphasizing the potential use of HbA1c measurement as a biomarker in the general population. Regarding risk stratification, HbA1c levels could be particularly important in subjects with HbA1c levels > 38.8 mmol/mol (5.7%), indicating a potential prediabetic metabolism and potential risk of cardiovascular disease. In addition, the findings might be of particular importance in individuals younger than 55 years who showed a more pronounced association of HbA1c levels and cardiovascular mortality. However, the cost-effectiveness of testing HbA1c levels in an asymptotic general population needs further evaluation.The present study employed one of the largest population-based datasets with predominantly harmonized data on HbA1c is necessary for cardiovascular risk assessment.Further research and external validation in a clinical setting are required to define whether an additional standardized measurement of HbAAdditional file 1. Online supplementary material.