External sulfate attack (ESA) of cementitious materials has been studied worldwide for a very long time. This physical/chemical interaction between sulfate ions and the cement hardened elements affects the long-term durability of concrete structures: cracking, spalling or strength loss of concrete structures. To study these damaging phenomena, some standardized and non-standardized accelerated aging tests are used to evaluate the performance of cements in sulfate-rich environments. However, these existing methods do not adequately predict field performance and some shortcomings or deficiencies still exist: change of degradation mechanisms when using high concentrations of sulfate, variable boundary conditions and small specimens compared to the real concrete structures. In this work, a critical review of some existing test methods and foreign national standard methods for ESA are presented, analyzed, and discussed. This results in some proposed recommendations for improving these methods to meet the needs of structure managers. External sulfate attack (ESA) is a complex concrete structural deterioration process that is initiated when sulfate present in the external environment penetrates into concrete ,2,3,4,5.Indeed, a large part of the complexity of this reaction stems from the variety of ions like calcium, sodium and magnesium sulfate that can be involved in the alteration of the paste composition . MoreoveAlthough the mechanisms associated with ESA remains a highly active and controversial research area, there is a need to build concrete infrastructures even in places where concrete is exposed to a sulfate-containing environment. In this context, the industry has outlined several blended cements with improved performance against ESA and several laboratory test methods have been developed to determine the sulfate resistance of these cement mixtures.Usual ESA testing methods involve the immersion of cement paste, mortar or concrete samples into a sulfate solution leading to the diffusion of sulfate ions through the samples. After several months to more than one year of immersion, such tests can be used to assess the change in the lengths and masses of samples induced by ESA in parallel through visual inspection ,24,25,26Moreover, the two accelerating methods of exposing samples to a high sulfate solution and high temperature do not represent the real conditions: the high concentration commonly used in the accelerated tests corresponds to about ten times the maximum values found in the field, and this could change the degradation mechanisms occurring in the concrete structures. Furthermore, the typical water temperature in the field range from 10 to 20 °C, which is significantly lower than that maintained during accelerated tests ,23,24,28In contrast to the lack of representativeness of the previous acceleration protocols, it is notable that the reduction in the curing time of a sample as an accelerating tool can correspond to a representative field condition considering that concrete structures can be exposed to external aggressive environments early in their construction. This early-age exposure has an obvious influence on the concrete strength and transfer properties, which are first-order parameters affecting the durability of the material.Current test methods have been criticized in several publications for their slowness or exposure conditions, such as pH and sulfate amount in immersion solutions ,30,31,32Accelerated test methods for evaluating the properties of cementitious materials in sulfate-rich environments such as the ASTM method  have recThere are several review papers in the literature of accelerated test methods applied to the study of ESA ,51,52,53This paper is organized as such: after this introduction, Several tests are available to study the different aspects of ESA. Some of the experimental methods replicate the process of this attack by immersing the samples in a test solution or by performing continuous wetting and drying cycles ,54,55,562 solution at 35 °C. After curing, the samples were immersed in a sodium sulfate solution (0.7 M) at 25 °C for 60 days. Hughes  als als84] aSeveral Chinese standards were established to prescribe ESA test methods ,89,90. TAs described above, GB 749-1965  is used 2SO4 solution should be maintained at around 7 throughout the test which can represent field conditions. Also, the Na2SO4 solutions were replaced every month to ensure that the experimental conditions are closer to the field conditions, in which mortar is exposed to a continuous supply of sulfate ions. This standard method allows calculation of a corrosion resistance coefficient that evaluates the ability of cementitious materials to resist ESA. An example of the result given by this method is highlighted in Finally, there is an update of the GB/T749-2001 test method that was published in 2008 (GB/T749-2008) . In this2+) associated with this test lead to a particular type of ESA that is mostly encountered in marine environments. However, the experimental protocol proposed by Messad [Various experimental works are listed in the literature as ways to accelerate ESA; however, the majority of the testing procedures require a long exposure time. For example, in the current French standard established for special products intended for hydraulic-binder-based concrete work , the tesy Messad  uses a hy Messad  are listIn the protocol proposed by Messad, both the cylindrical (110 mm in diameter and 220 mm in height) and prismatic (7 cm × 7 cm × 28 cm) specimens were demolded and placed directly in water for 28 days. At the end of this phase, the concrete samples were subject to a drying phase (4 weeks at 60 °C) before being saturated under vacuum with a high-concentration sulfate solution (8.9 g/L sodium sulfate). This pre-saturation cycle, which lasted 48 h, can be considered a means of accelerating the test by reducing the time needed for a sufficient amount of sulfate ions to diffuse inside the sample. This step helped to overcome the kinetics of the penetration of sulfate ions linked to the physical resistance of concrete and was, therefore, an essential factor in the acceleration of the attack . At the Most of the accelerated-aging methods developed for the study of ESA in cement paste, mortar or even concrete samples use high sulfate concentrations of the order of 10 g/L or more. This order is larger than that encountered in real life situations and on sites . This hi3; 2 × 2 × 16 cm3; 4 × 4 × 16 cm3 and 7 × 7 × 28 cm3) were tested to investigate the effect of size on their performance against ESA. The prisms were placed at 100% RH for 24 h and then left under limewater for 27 days. After the curing period, the mortar samples were immersed in a 3 g/L sodium sulfate solution. From the expansion measurements . During the first phase (latency period), the expansion was stable and low. However, the second phase was characterized by an important increase in expansion for all samples. Interestingly, in the case of smaller prisms (1 × 1 × 10 cm3 and 2 × 2 × 16 cm3), the second phase started early (after 200 days of immersion in sodium sulfate solution) whereas significant expansion was found in the 4 × 4 × 16 cm3 prisms after 400 days of immersion [In the experimental program used by El-Hachem et al. , mortar ents see , two phammersion .3) in three different sodium sulfate solutions with the following concentrations: 3 g/L, 10 g/L, and 30 g/L. The curing phase in limewater lasted for 15 days. The experimental results (see 42−) [Moreover, the effect of the sulfate concentration of the immersion solution was evaluated by fully immersing mortar prisms (2 × 2 × 16 cmults see  clearly see 42−) .This experimental study validated that both the size of the sample and the concentration of the solution can influence the behavior of a cementitious material exposed to ESA. Based on the results, the exposure of small mortar samples to high concentrations of sodium sulfate leads to greater degradation within a short period of time.In Germany, studies on effects of sulfate attack on cement-based materials are conducted by applying one of the three accelerating testing methods : the SVAMielich and Ottl  conducte2 solution, immediately followed by 91-days of immersion in high sodium sulfate solution (29.8 g/L). Prisms were fabricated with two cement types (CEM I/OPC and CEM III/B with 73.8% fly ash) and two w/c ratios (0.5 and 0.68). The higher w/c ratio was only used in the casting of CEM III/B prisms. Hence, three different mortar mixes were finally tested against accelerated sulfate attack . The resistance of mortar samples was investigated by monitoring the expansion at the initial state (after cure in saturated Ca(OH)2 and therefore before any contact with the sodium sulfate solution) and during the accelerated attack. Expansions were measured at specific time intervals . The study conducted by Mielich and Ottl  was compIt can be observed that the expansion of mixture 2 and mixture 3 samples (both made with CEM III/B) did not exceed the recommended criterion value of 0.5 mm/m at the end of the exposure period. However, mixture 1 containing CEM I showed significant expansion after 91 days of immersion in the sodium sulfate solution that exceeded the recommended threshold limit. According to Mielich and Ottl  these ob3 cubes made of three different concrete mixtures  were cast and kept in a moist room at 20 °C and 95% of RH for two days. After 48 h, cores with a 50 mm diameter were extracted from these lab-manufactured specimens, sawed (at 150 mm length) and polished at the top in order to resemble the site cores as much as possible. Before immersion in the sodium sulfate solution (29.8 g/L) at two different temperatures (20 °C and 6 °C), all concrete specimens (extracted from the site and from cubes) were stored for 12 consecutive days at 20 °C and 65% RH. A temperature of 6 °C was considered in order to investigate the thaumasite form of sulfate attack. The expansions measured during the sulfate attack are shown in In the second part of the study, concrete cores with a diameter of 50 mm that were extracted from the foundation of a damaged structure were tested. The concrete of the building was produced with CEM III/B containing 50% fly ash and with a high w/c ratio of 0.68 (this concrete mixture is labeled S IV in the following). To produce cored companion samples, 200 mmFrom 3) made from various types of cement  were cast into special molds containing, in the center part, three prismatic locations for mortar with two containers on the sides for the sulfate solution (as shown in 2SO4) solution, while a 2.8% NaOH solution filled the anodic part of the testing mold. One of the surfaces of the specimen was exposed to sulfate solution (40 mm × 40 mm), while the remaining five surfaces were sealed with Vaseline  and (2.7%) at a depth of 0.2 cm after 30 days of SA + EF and 90 days of SA + EF, respectively [In another experimental program, the same group of researchers applied  a modifiB/T 2008  by chemiectively . These rIn addition, the mechanical properties of the attacked samples  were evaluated. To identify the changes in the microstructure and the mineral phases at the end of the accelerated attack, microscopic observations were carried out using a scanning electron microscope and X-ray diffraction techniques. In conclusion, it was observed that accelerating ESA via the electrical pulse technique helped, not only by increasing the migration of sulfate ions into the samples but also by accelerating the leaching process which led to a complete dissolution of portlandite (CH), followed by a decomposition of C-S-H. The overall increase in the amount of sulfate diffused into the mortar samples induced severe deterioration and damage by the formation of both ettringite and gypsum. This was confirmed by the mechanical strength losses observed after the attack. These results show that this accelerating technique, if properly applied, can be considered a fast method that can be used to study the effects of ESA on cement-based materials ,102,104.As previously discussed, several methods are used to determine the sulfate resistance of cement-based materials. Most test methods involve full or partial immersion of mortar, concrete, or cement paste in a sulfate solution with a specific concentration and with or without pH regulation ,30,46,472SO4 [2SO4, while monitoring the pH using a pH controller. During their experimental work, the surrounding sulfate solution was purely acidic stabilizing the pH at 6.2. The results showed that, under such exposure conditions, the degradation process was mainly dominated by gypsum formation. Moreover, it has been reported that, under these conditions, the low C3A content did not improve the resistance against sulfate ingress because negligible amounts of ettringite are formed during ESA compared to gypsum [The pH of sulfate solution can significantly affect the progress of ESA. The leaching process that occurs during erosion and the dissolution of the cement paste phases directly affects the pH of the solution. Some studies recommend the use of pH regulation while others prefer to maintain uncontrolled pH. For example, the method developed by the GeM , which, 2SO4 . Moreove2SO4 ,115 trieo gypsum ,115.2+) leaching and crack formation at the surface level [In ,30, the ce level . Howeverce level .3), leads to faster results compared to the standard 25 × 25 × 285 mm3 specimens [3) placed in contact with a high concentration sodium sulfate solution (50 g/L). In a study by Ferraris et al. [3). Interestingly, the same level of degradation is achieved but in a shorter time. Based on this, Ferraris et al. stated that the use of small samples could reduce the time needed to observe critical damage caused by ESA.Studies on the effects of the specimen size and/or shape have shown that a higher expansion rate with smaller prisms  and cylinders with varying diameters  and lengths of 152 mm. The results were almost similar for both geometries, confirming the importance of size over shape (geometry) when accelerating ESA.In the same study, Ferraris et al. tried to cover not only the effect of size but also the effect of shape (prism or cylinder). They compared the expansion evolution between prisms  or to the low temperatures which enhance the formation of thaumasite by attacking the C-S-H ,106,114.Most laboratory tests provide a chronological evaluation of the samples. However, the deterioration state can be immediate in field tests, and this can be related to the difficulty of controlling the exposure conditions, such as the temperature and relative humidity, in the field. The comparison between exposure to sulfate under controlled laboratory conditions and in the field has shown good correlations in terms of distress and failure. However, the possibility of reproducing the technology used in the ESA study and transferring it to a construction project is uncertain. Concrete quality assessment methods at real construction sites primarily focus on non-destructive testing to characterize the structure in a particular condition. With all the problems and complications associated with ESA it is doubtful whether a single test can solve all these problems. In addition, investigating all potential exposures and associated conditions is extremely expensive and time-consuming. To overcome these difficulties, several authors have performed thermodynamic calculations with the help of specific software to model and predict the progress of the ESA by taking into consideration several materials placed in different erosive solutions at different temperatures ,32,118.Many researchers believe that most of the test methods listed in literature evaluate the exposure of mortar or paste samples to sodium sulfate solutions within unreasonable time frames . HoweverThe accelerated ESA protocol intentionally induces microcracks in the experimental material resulting in greater expansion due to specific sulfate concentration and pH regulation (LMDC and GeM methods), and has been shown to be effective, but time consuming. The methods developed in Europe, when compared to the US methods recommended by ASTM or USBR, are considered more effective in the long term with a significant increase in the rate of sulfate ingress, resulting in significantly faster rates of expansion and deterioration ,119.Accelerated tests on mortar samples were proven to be significantly shorter in time compared with tests performed on concrete specimens. This is mainly attributed to the specimen size, its permeability, and the cement content. However, using concrete with a relatively high w/c ratio was associated with significant improvements, obtaining results over a shorter period depending on the type of mixture and mineral additions ,122,123.Several attempts have been made to develop a test method that can quantify the distress and deterioration levels caused by ESA in construction projects. However, it should be noted that the kinetics of the in situ attack are much slower than in accelerated tests, so it may take several years to see serious deteriorations of the investigated structure. This raises concerns about the possibility of finding a way to analyze the entire attack mechanism. Additionally, the application of a testing method to existing projects or structures requires several field inspections and appropriately developed techniques, which can be complex and expensive for construction firms. Therefore, the results obtained in laboratory tests are considered crucial to determine the possible results and consequences of exposure of concrete structures to ESA . AccelerBased on this, results on concrete are generally considered more representative of the cases commonly seen in fields . It shouEach experimental program devoted to the study of ESA has different strengths and weaknesses. Some researchers believe that the use of higher sulfate concentrations is the most efficient way to achieve adequate results in an acceptable amount of time. However, others state that this technique can only approximate the natural process of the attack. Concerning the shape  and the type  of samples, the debate is always framed in terms of the reliability and representativeness. This highlights the need to unify all these propositions in a common experimental approach. However, the difficulties associated with ESA make any future work subject to many debatable interpretations. Lastly, it is recommended that further studies should be conducted in order to develop well-established scientific methods that are directly related to real-life conditions and exposure sites, with the definitive goal of benchmarking accelerated controlled laboratory tests. Combining predictive models and software calculations with rapid metric testing will ultimately yield useful solutions, but further research is needed to implement them into standards and building codes.-) are partially absorbed by calcium silicate hydrate (C-S-H) and can connect to monosulfoaluminate to form Friedel’s salts or hydrocalumite (3CaO Al2O3 CaCl2 10H2O) which can decrease the effects of ESA by limiting the stability of ettringite. Due to the complexity of the process, the penetration of chlorides and sulfates into cement-based material exposed to marine environments is studied independently.An appropriate test method for studying ESA resistance should be reliable and reproducible, but also rapid, relevant and practical, and should not exceed expectations. However, it should be considered that although tests may address certain performance and durability issues for general industrial or scientific issues, they should relate to actual and situational conditions as closely as possible. This is not the case for most external sulfate resistance testing methods which only measure the resistance of a cementitious binder  when fully immersed in a sulfate solution . In Sulfate attack is a widespread form of physical–chemical attack on concrete physical-chemical attack and has always been an important part of research on the durability of concrete structures. Although various testing methods for resistance to sulfate attack have been proposed, so far each method has certain advantages and disadvantages which should be considered in terms of ease of implementation and cost representativeness. With certain limitations, no method can quickly and accurately reveal the mechanism associated with concrete sulfate attack, especially for the long-term prediction of various properties of concrete in sulfate environments. It is therefore particularly important to continue research efforts to develop optimized sulfate attack test methods.The ASTM C 452-15 can result in the sulfate attack of anhydrous compounds, and the presence of gypsum can cause sulfate attack in the fresh state. This early attack means that the testing conditions of this method are not similar to field exposure.The ASTM C 1012 is a much longer test method (about 18 months) compared to the ASTM C 452, and it is not considered to be the best or the most connected to real-life exposure conditions.The USBR 4908 method is also a much longer test as the test period varies between 1 and 2 years. This method does not support the continuous development of new sulfate-resistant cements.The Swiss standard SN 505262/1 can provide relevant information about the resistance of concrete against ESA. However, the exposure conditions of the specimens are not similar to the field conditions (consecutive cycles of drying–wetting followed by full immersion of the specimens).The Canadian standard CSA A3004-13 is very similar to the ASTM C 1012 method, but it considers the formation of ettringite and thaumasite forms of the sulfate attack. This method uses a very highly concentrated sulfate solution that is not consistent with the field conditions.The Asian standard GB 749-1965 requires a long test period without any information about the sulfate solution concentrations. Even though the duration is shorter than that of other proposed standards , this method has a major disadvantage in that the size of the test specimen is too small, and no complementary tests have been proposed to explain the test results.In this paper the main methods of evaluating mortar and concrete resistance to sulfate attack are reviewed. Some limitations of these methods are mentioned below:Non-standardized methods were also proposed in this paper. All of them are satisfactory but could conceivably benefit from improvements . The continued research and efforts in the field of sulfate attacks have aided in the exploration of new techniques  that could be used to study ESA over short periods of time. However, many researchers believe that these methods modify the ESA mechanisms encountered in reality. In this context, another question may arise in terms of the ability of these accelerated techniques to reflect realistic and achievable conclusions regarding ESA resistance. When a cementitious material in the field is exposed to a sulfate-rich environment, it would be of paramount interest to test the resistance of its composition beforehand. In addition, important factors related to the type of sulfate environment, sulfate solubility, and concentration should be determined and evaluated during the experimental approach. For example, having both sodium and magnesium sulfates makes the mechanism of ESA more complicated, and in this case, the traditional experimental approaches of immersion in a sodium sulfate solution (US and European methods) become inefficient for studying the effects of the coupling between sodium and magnesium. Moreover, thaumasite formation and salt crystallization are two independent types of attack that require the use of different experimental approaches to analyze the damage process.


Drosophila Kc cells. In vitro experiments demonstrate that JH-induced phosphorylation sites in the basic helix-loop-helix (bHLH) domain, but not in the Per-Arnt-Sim-B (PAS-B) domain, are required for maximization of Met transcriptional activity. Moreover, phosphoproteomics analysis reveale that JH also induces the phosphorylation of Hsp83, a chaperone protein involved in JH-activated Met nuclear import. The JH-induced Hsp83 phosphorylation at S219 facilitates Hsp83-Met binding, thus promoting Met nuclear import and its transcription. By using proteomics, subcellular distribution, and co-immunoprecipitation approaches, we further characterized 14-3-3 proteins as negative regulators of Met nuclear import through physical interaction with Hsp83. These results show that JH membrane signaling induces phosphorylation of the key components in JH intracellular signaling, such as Met and Hsp83, and consequently facilitating JH intracellular signaling.Juvenile hormone (JH) regulates insect development and reproduction through both intracellular and membrane signaling, and the two pathways might crosstalk with each other. Recent studies have reported that JH membrane signaling induces phosphorylation of the JH intracellular receptor Met, thus enhancing its transcriptional activity. To gain more insights into JH-induced Met phosphorylation, we here performed phosphoproteomics to identify potential phosphorylation sites of Met and its paralog Germ-cell expressed (Gce) in  Drosophila melanogaster  (Juvenile hormone (JH), which is primarily produced and secreted by the corpus allatum (CA) in insects, is crucial for regulating metamorphosis and reproduction. Methoprene-tolerant (Met), a transcription factor belonging to the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family, was first identified in nogaster . Howeverreceptor . A paralreceptor . Both Mein vitro . Importaansition , exhibitansition . The reqosophila . In the osophila . Then, Mosophila . The cha (Kr-h1) . Nucleop (Kr-h1) .in vivo and in vitro experiments indicate that JH membrane signaling is able to activate RTK -PLC-PKC pathway independent on JH intracellular signaling  is proposed to phosphorylate Met and Tai, and enhance Met-Tai DNA binding activity with JH response elements (JHRE)  . Additios (JHRE) . In Heliwith Tai . HoweverDrosophila and would be helpful to advance our understanding of the complex JH signaling network.To address these issues, we detected multiple phosphorylation sites of Met and Gce after methoprene treatment with a biotinylation tagging method and found that phosphorylation sites in the bHLH domain are important for transcriptional activity of Met. Second, JH membrane pathway phosphorylated Hsp83 at S219 to promote Met nuclear import and enhance JH intracellular signaling. Finally, 14-3-3 proteins recognized Hsp83 that seemed acting as a bridge between Met/Gce and 14-3-3 proteins, and subsequently sequester Met in the cytoplasm to inhibit its nuclear import. This work provides novel insights into JH signaling transduction in Drosophila Met (NM_078571) and Gce (NM_078605) open reading frame was amplified from the larval fat body cDNA library, and BirA (NC_000913.3) was amplified from E. coli. All the PCR products were cloned into the pClone007 vector . BirA was then inserted into the pUAST vector between the EcoRI and XbaI sites to generate a control construct expressing only the BirA protein. For the generation of Met and Gce overexpression lines, vectors were constructed with the Gibson assembly method. First, the pUAST vector was digested by EcoRI/XbaI. The P2A-BirA sequence was amplified from the pClone007-BirA vector. When a P2A peptide (ATNFSLLKQAGDVEENPGP) was added to the N-terminus of BirA, 5 amino acids (GGSGS) were added to the N-terminus, and 3 amino acids (GGS) were added to the C-terminus of the P2A peptide as a linker. The FLAG tag (DYKDDDDK) and an Avi tag (GLNDIFEAQKIEWHE) were added to the 5′ end by reamplifying the above PCR products with FLAG-Avi-P2A-F oligos and the BirA-R oligo. PCR of the coding sequence of Met or Gce from the pClone007-Met or pClone007-Gce vector was performed, and then we used a Hieff Clone Plus Multi One Step Cloning Kit  for assembly. Finally, we obtained the pUAST-Met-4 × FLAG-Avi-P2A-BirA (UAS-Met-BirA) or pUAST-Gce-3 × FLAG-Avi-P2A-BirA (UAS-Gce-BirA) vector, in which BirA was fused at the C-terminus. Meanwhile, we also constructed pUAST-BirA-P2A-Met-3 × FLAG-Avi and pUAST-BirA-P2A-Gce-3 × FLAG-Avi, with BirA fused at the N-terminus, using a similar method to assess the translational efficiency of different termini.The full-length open reading frame of Met, Gce, Hsp83 (NP_523899), 14-3-3ε (NM_169796) and 14-3-3ζ (NM_001273916) were amplified. Met-FLAG, Met-bHLH+(amino acids 1–97)-FLAG, Met-PAS-A+(amino acids 88–196)-FLAG, Met-PAS-B+(amino acids 187–716)-FLAG, Met-PAS-B (amino acids 403–510)-FLAG, Met-PAS-B + T265A-FLAG, Met-PAS-B + S531A-FLAG, Met-PAS-B + T535A-FLAG, Met-PAS-B + S652A-FLAG, Met-PAS-B + T265A/S531A/T535A/S652A-FLAG (4 m), Met-4m-FLAG, MetS256A/T265A/S531A/T535A/S651A/S652A-FLAG (6 m), Gce-FLAG, Gce-bHLH+(amino acids 1–335)-FLAG, Gce-PAS-A+(amino acids 326–415)-FLAG, Gce-PAS-B+(amino acids 406–956)-FLAG, Hsp83-FLAG, Hsp83-V5, Hsp83S219A-V5, 14-3-3ε-HA, and 14-3-3ζ-HA were separately generated and constructed into the pUAST vector for overexpression in Kc cells.Moreover, the full-length cDNAs of Drosophila Kc cells were cultured in Schneider’s medium. UAS-BirA, UAS-Met-BirA, UAS-Gce-BirA or both was co-transfected with the Act5c-Gal4 vector into cells using Effectene transfection reagents  according to the manufacturer’s instructions. After 47 h, the old medium was replaced with fresh medium containing 10 μM methoprene  and then incubated for an additional 1 h. The harvested cells were lysed in ice-cold NP-40 lysis buffer  supplemented with protease and phosphatase inhibitor cocktail  and protease inhibitor cocktail  for western blotting and immunoprecipitation. Immunoprecipitation was performed overnight at 4°C with streptavidin beads  and was followed by three 15 min washes using lysis buffer supplemented with protease and phosphatase inhibitor cocktail and protease inhibitor cocktail. After washing, the purified proteins were eluted by competitive elution and boiling elution. Competitive elution was eluted with free biotin or with an equal bead volume of SDS sample buffer containing dithiothreitol (DTT) at 95°C for 5 min, boiling elution was eluted by boiling bead after adding protein loading buffer. For phosphorylation site mapping, Met or Gce proteins were resolved by 10% SDS-PAGE, and the excised bands were used for protease digestion and mass spectrometry.After protease digestion, peptides were dissolved in 0.1% formic acid (FA) and 2% acetonitrile (ACN) and separated by a reversed-phase analytical column . LC separation of the peptides was initiated with an increasing gradient from 5 to 50% solvent B (0.1% FA in 80% ACN) over 20 min, and ramped to 90% for 10 min, followed by a 5 min holding. All steps were performed at a constant flow of 300 nL/min. MS analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). Intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were selected for MS/MS using a normalized collision energy (NCE) setting of 27, and ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 30 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 1E5 ions were accumulated for generation of MS/MS spectra. For MS scans, the scan range was 350–1800 m/z.D. melanogaster Reference Sequences database . The parameters were as follows: trypsin (full), 2 maximum missed cleavages, cysteine carbamidomethylation (C) as the fixed modification, and methionine oxidation as the variable modification. For phosphorylation site mapping, phosphorylation of serine/threonine/tyrosine was added as another variable modification. The precursor ion mass tolerance in the initial search was 20 ppm. The results were filtered according to a 1% false discovery rate (FDR) at the peptide and protein levels.Protein identification was performed with MASCOT software by searching the UniProt UAS-Hsp83-V5 was co-transfected with the Act5c-Gal4 vector into cells using Effectene transfection reagents according to the manufacturer’s instructions. After 46 h, the old medium was replaced with fresh medium containing 10 μM methoprene, and the cells were incubated for an additional 2 h. The harvested cells were lysed in ice-cold NP-40 lysis buffer (supplemented with protease and phosphatase inhibitor cocktail and protease inhibitor cocktail) for immunoprecipitation. The lysates were incubated with V5 antibody for 4 h and then with Pierce protein A/G agarose  overnight at 4°C. Beads were collected by slow centrifugation, washed 4 times with lysis buffer and resolved by 10% SDS-PAGE. The excised bands were used for protease digestion and mass spectrometry.6, and the maximum ion injection time was 20 ms. The top 20 most abundant precursors in the full scan were selected, fragmented by higher-energy collisional dissociation and analyzed via MS/MS with a resolution of 15,000, an AGC target value of 1 × 105, a maximum ion injection time of 45 ms, a normalized collision energy of 27%, an intensity threshold of 2.2 × 104, and a dynamic exclusion time of 20 s.After protease digestion, the peptides were separated in a C18 analytical column (15 m × 150 μm × 1.9 μm). The gradient comprised a decrease from 94 to 0% mobile phase A (0.1% FA) and an increase from 6 to 100% mobile phase B (0.1% FA in 80% ACN) over 25 min and then a hold at 0% mobile phase A and 100% mobile phase B for 5 min. The separated peptides were analyzed with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The full scan range was from 350 to 1,500 m/z, the resolution was 60,000, the automatic gain control target value was 3 × 10D. melanogaster Reference Sequences database . The search parameters were set as follows: carbamidomethyl was specified in PD 2.2 as a fixed modification, oxidation of methionine and acetylation of the N-terminus were specified in PD 2.2 as variable modifications, serine/threonine/tyrosine phosphorylation was another variable modification, the mass tolerance for precursor ions was 10 ppm and a maximum of 2 missed cleavage sites were allowed. The results were filtered to a 1% FDR at the peptide and protein levels.Protein identification was performed with Proteome Discoverer 2.2  by searching the UniProt Western blotting was performed as previously described . In brie2D gel electrophoresis was performed as previously described . Total pg for 15 min at 4°C. The lysates were incubated with a FLAG, V5, or HA antibody for 4 h and then with Pierce protein A/G agarose overnight at 4°C. Beads were collected by slow centrifugation, washed 4 times with lysis buffer and analyzed by SDS-PAGE followed by western blotting.Forty-six hours following transfection, 10 μM methoprene  or DMSO was added to Kc cells for 2 h. The cells were then washed with PBS and harvested and lysed in ice-cold NP-40 lysis buffer containing protease and phosphatase inhibitor cocktail and protease inhibitor cocktail. The extracts were clarified by centrifugation at 12000 × T265A-FLAG, Met-PAS-B + S531A-FLAG, Met-PAS-B + T535A-FLAG, Met-PAS-B + S652A-FLAG Met-PAS-B+4m-FLAG, Gce-bHLH + -FLAG, Gce-PAS-A + -FLAG, Gce-PAS-B + -FLAG, Met-4m-FLAG, Met-6m-FLAG, 14-3-3ε-HA, and 14-3-3ζ-HA was measured by immunohistochemistry as described previously . All qRT-PCR was performed with at least three biological replicates, each with triplicates, by using Hieff qPCR SYBR Green Master Mix  on a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). The final volume of each reaction was 20 μL, and the PCR thermocycling was initiated from 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. After the PCR amplification, a melting program was also included to verify the specificity of the products. Relative expressions of the target genes were calculated by the 2pGL3-JHRR  followed by Duncan multiple comparisons test, respectively, at the level of p < 0.05.Unless otherwise specified, all the data subjected to statistics are mean ± standard deviations from independent replicates, and the statistics was analyzed with IBM SPSS Statistics 19.0. Significant differences between two groups and among multi-treatments were determined with the Student’s Drosophila Kc cells. In response to JHA, the phosphorylation of both proteins in was elevated at 2–4 h, and the elevation was sustained for 6 h . We generated vectors that express Met-BirA or Gce-BirA, a synthetic linear construct with an added biotin ligase (BirA) molecule at the C-terminus -treated cells. To assess the translational efficiency of different termini, we initially tested Met or Gce protein levels in Kc cells. We found that Met and Gce protein levels were higher when BirA was fused at the C-terminus than that at the N-terminus , PAS-A+ (amino acids 88–196) and PAS-B+ (amino acids 187–716)  . Compare187–716) . By cont187–716) . Likewis187–716) . Given tKr-h1 transcriptional level was similar to that in intact Met-overexpressing cells after JHA exposure  or threonine (T) to alanine (A) and generated 5 phosphor-inactivated PAS-B+ mutant constructs. All these mutant forms failed to prevent their subcellular trafficking in the presence of JHA . Likewisexposure .Kr-h1 transcriptional level was significantly higher in Met-overexpressing cells treated with JH than in Met-6m-overexpressing cells , and Hsp83, are indispensable for modulating JH action or Met nuclear import . To deteWhile 14-3-3 proteins have mostly been reported to specifically interact with phosphoserine-containing motifs such as RSXpSXP (mode I) and RXXXpSXP (mode II) in their ligands , some noWe next asked whether there exists a protein(s) acting as molecular bridge between Met/Gce and 14-3-3 proteins. As an important partner protein of Met, Hsp83 is required for Met nuclear import . We therKr-h1 expression, whereas 14-3-3 transcript levels remained unchanged (Kr-h1 expression or JH response region (JHRR) binding activity in Kc cells exposed to JHA  and Met27gce2.5K (lacking JH intracellular receptors) cells  cells . This fis) cells  predictes) cells . Importas) cells , the equ complex .In conclusion, this study reveals novel effects of JH membrane signaling on the transduction of JH intercellular signaling. On the one hand, JH membrane signaling potentiates the DNA-binding activity of the bHLH domain of Met. On the other hand, JH membrane signaling promotes the interaction between Hsp83 and Met by phosphorylating Hsp83 at S219. Finally, phosphorylation of Hsp83 modulates the JH response to enhance the transcriptional activity of Met, whereas 14-3-3 proteins negatively regulate JH action by physically interacting with Hsp83 . This st


This article presents state-of-the-art research regarding fibrous concretes produced with wollastonite, such as mortars, conventional concrete, engineered cementitious composites, geopolymer concrete, self-compacting concrete, ultra-high-performance concrete and pavement concrete. The use of synthetic wollastonite, which is a novel issue, its high aspect ratio and allowing the use of waste material are also evaluated. Studies in the literature show that the use of wollastonite in different types of concrete improves performance properties, such as mechanical/durability properties, and provides environmental–economic efficiency. It has been proven by studies that wollastonite is a material with an inert structure, and, therefore, its behavior is similar to that of a fiber in cementitious composites due to its acicular particle structure.Production of cement and aggregate used in cement-based composites causes many environmental and energy problems. Decreasing the usage of cement and aggregate is a crucial and currently relevant challenge to provide sustainability. Inert materials can also be used instead of cement and aggregates, similar to pozzolanic materials, and they have positive effects on cement-based composites. One of the inert materials used in cement-based composites is wollastonite (calcium metasilicate-CaSiO Beca in 2020 , along w in 2020 .2, has a molecular weight of 116.2 [2O3, Fe2O3, MgO and Na2O could also be available in the chemical composition of wollastonite. Wollastonite is also called calcium metasilicate, and the chemical formula of this mineral is CaSiO3 [One of the most used inert materials is wollastonite. Wollastonite was discovered in 1886 by British chemist Sir Wollaston and was named after him ,15. Wollof 116.2 . The ches CaSiO3 ,19,20,21s CaSiO3 . Wollasts CaSiO3 ,23,24,25s CaSiO3 ,27,28. Ms CaSiO3 , and it s CaSiO3 ; thus, rs CaSiO3 ,29.Wollastonite can be artificially synthesized with different materials. For example, it was produced with eggshell and silica , rice huIn recent years, many studies have been conducted on the use of both natural wollastonite and synthetic wollastonite in cement-based composites. Especially, the use of synthetic wollastonite is novel and contains valuable findings for future studies regarding cement-based composites because of the high aspect ratios of acicular particle structure. In this study, use of wollastonite in mortar, conventional concrete, engineered cementitious composites, geopolymer concrete, self-compacting concrete, ultra-high-performance concrete and pavement concrete was investigated. The effects of wollastonite usage in different cementitious composites on the physical, mechanical and durability properties were presented and discussed. In addition, general evaluations were conducted according to these discussions. Finally, suggestions were put forward regarding possible future research directions.Cement paste consists of cement and water; mortar consists of cement, water and fine aggregate. Studies on cement paste or mortar were performed in the form of using wollastonite replacement of sand and/or cement. Doner et al.  used 5% In some studies, comparisons were performed by using wollastonite and an additional material. In a study in which the effect of wollastonite average particle size was taken into account, 3.5 µm and 9.0 µm average particle size wollastonite was used at a rate of 10%, 20%, 30%, 40% and 50% by mass in replacement of cement . In addi3 tended to decrease with the addition of synthetic wollastonite. Öz and Güneş [In addition, studies on the use of synthetic wollastonite on mortars were also carried out. Kalkan et al.  used 0%,nd Güneş , on the nd Güneş ,20,32,39nd Güneş  12% syntnd Güneş ; this diConcrete is formed by mixing cement, water, coarse aggregate, fine aggregate, chemical additives and mineral additives at a certain rate. The usage of wollastonite in concrete was performed in three different ways: replacement of sand, cement and their combination. Kh  used 10%In some studies for concrete, different material combinations were attempted, including wollastonite replacement of cement or replacement of cement+fine aggregate. Kalla et al.  examinedEngineered cementitious composite (ECC) is defined as a special type of high-performance fiber concrete and shows strain hardening similar to a ductile material after the first crack and has approximately 300 to 500 times greater tensile strain capacity compared to conventional concretes. In a study conducted for ECC, synthetic wollastonites with different high aspect ratios were used instead of cement, fly ash and cement + fly ash . The minGeopolymer concrete is formed as a result of geopolymerization of aluminosilicate source materials  with sodium- or potassium-sourced alkali activators ,46. WollIn a study where wollastonite was used as a reinforcement, different material combinations were also tested in addition to wollastonite . SubstitSelf-compacting concrete is a concrete that can be compacted without requiring any labor and fills the formwork homogeneously thanks to its self-flowing feature . Jindal 2 reacts with SiO2 in the wollastonite microfibers and forms C–S–H [Ultra-high-performance concrete (UHPC) is defined as a type of concrete with very high strength and durability. Kwon et al.  used wolms C–S–H ,57. The ms C–S–H . These fms C–S–H . An incrms C–S–H  examinedms C–S–H  formed tPavement is the top layer of the superstructure and forms a smooth rolling surface for vehicles. Due to the positive effect of wollastonite on the performance properties of concrete, studies were carried out on its use in pavement. Ransinchung and Kumar  used wolWollastonite uses and different effects are summarized in 2). Wollastonite  has been Investigated by many researchers for approximately the last two decades. Moreover, artificially synthesized wollastonite is a novel and high-quality inert material because of the higher aspect ratio and the possibility of production by using different materials. This study systematically presented the use of natural and synthetic wollastonite in cement-based composites. The use and effects of wollastonite in cement-based composites are as follows:Decreasing the usage of cement and aggregate is a crucial and currently relevant challenge. As an alternative to cement and aggregate, pozzolanic materials have been used for many years and have positive effects on cement-based composites. Not only pozzolanic materials but also inert materials can be used instead of cement and aggregate. One of the inert materials used in cement-based composites is wollastonite, which has two main basic components  are further enhanced with wollastonite, and 6% wollastonite substitution was stated as the optimum ratio. In addition, synthetic wollastonite with high aspect ratios of 44:1 and 33:1 was tested. The synthetic wollastonite with an aspect ratio of 44:1 showed better performance than 33:1. The effect of high aspect ratio was observed on ECC.Wollastonite was used instead of a precursor and sand in geopolymer concrete. The low mechanical property disadvantage of geopolymer was partially eliminated with the utilization of wollastonite. Strength increases were achieved with the use of 10–20% instead of sand.Positive performance properties were obtained from self-compacting concrete produced with wollastonite, both alone and in combination with different materials, up to 30% replacement.In ultra-high-performance concrete, the usage of wollastonite 27% instead of sand and up to 12% instead of cement increased the performance properties. Wollastonite can also increase sustainability without losing its performance properties.Only wollastonite and different material combinations were tested in pavements. Positive effects were observed in the use of up to 15% wollastonite.It has been proven by studies that wollastonite is a material with an inert structure, and, therefore, it behaves similar to a fiber in cementitious composites due to its acicular particle structure. The use of wollastonite in cement-based composites both improves mechanical-durability properties and reduces the use of cement and aggregate, therefore providing more sustainable production. Especially regarding synthetic wollastonite’s high aspect ratio and different material usage possibilities, its use up to 10% instead of cement is promising. In future studies, some durability tests  of using wollastonite can be performed for all cementitious composites. The effect of synthetic wollastonite on different cement-based composites (except mortar and ECC) and use of synthetic wollastonite instead of sand can be studied. Moreover, the life cycle assessment of cementitious composites produced with synthetic wollastonite can be investigated. Finally, synthetic wollastonite and different pozzolanic material combinations can be tested.


MAT1A mutations that are not yet well understood. Of the 4,065,644 neonates screened between November 2010 and December 2021, 35 individuals have been diagnosed with an estimated incidence of 1: 116,161 by a cutoff value of methionine 82.7 μmol/L and follow-up over 11 years. MATD patients with autosomal recessive (AR) type had higher clinical and genetic heterogeneity than those with autosomal dominant (AD) type. Fifteen unrelated AD patients harbored one well-known dominant variant, c.791 G>A or c.776 C>T, and were clinically unaffected with a mean plasma methionine (Met) value <300 μmol/L. Twenty AR cases have unique genotypes and presented a wide range of clinical abnormalities from asymptomatic to white matter lesions. Of them, 10 AR patients displayed severe manifestations, such as verbal difficulty, motor delay, development delay, and white matter lesions, with mean Met >500 μmol/L and thereby were treated with a methionine-restricted diet alone or in combination with betaine, folate, or vitamin B6, and were healthy finally. Neurological abnormalities were evidenced in two patients (P16 and P27) with Met values >800 μmol/L by MRI scan. Neurological abnormalities were reversed here by liver transplantation or by the determination of S-adenosylmethionine supplementation. Additionally, 38 variants of MAT1A were distributed within patients and carriers, of which 24 were novel and mostly predicted to be damaged. Our findings with an extensive clinical and genetic dataset provided new insights into its diagnosis and treatment and will be helpful for its optimal management in the future.Methionine adenosyltransferase deficiency (MATD) is a rare metabolic disorder caused by mono- or biallelic  MAT1A variants have been identified, respectively  is a rare metabolic disorder resulting in methionine adenosyltransferase (MAT) deficiency, with isolated hypermethioninemia and a low level of hepatic S-adenosylmethionine (SAMe). MATD can be transmitted either as autosomal dominant (AD) or autosomal recessive (AR) traits in which mono- or biallelic ectively . Althougectively , acroderectively , or evenectively . To dateectively . To advaectively . NeverthAll patients with a diagnosis of MATD and carriers based on newborn screening (NBS) program, genetic testing, and follow-up between November 2010 and December 2021 in Zhejiang Neonatal Screening Center, in which a total of 4,065,644 neonates were screened covering 364 maternity units in 90 counties. All procedures followed were in accordance with approval from the Research Ethics Committees, the Children’s Hospital of Zhejiang University School of Medicine. Patient information was tabulated without individual identifiers.Tandem mass spectrometry (MS/MS) was used to detect the concentrations of amino acids and acylcarnitine with dry-spot-blood samples in our NBS center. The reference value of methionine (Met) is 7.18–41.35 μmol/L and the reference range of Phenylalanine (Phe) was 23.3–100 μmol/L. Based on NBS screening data, the cut-off value for Met was set to 82.7 μmol/L, an upper limit of normal values in the 99.9th centile healthy newborns. The cut-off value for the ratio of Met/Phe was 1.1. Plasma total homocysteine (tHcy) was considered to be elevated at a concentration above 15 μmol/L. Newborns were recalled due to a higher Met concentration of more than 82.7 μmol/L alone or due to a higher ratio of Met/Phe. If the second test was also positive (Met >82.7 μmol/L or Met/Phe >1.1), the newborn was referred for a gene test. Cases associated with cystathionine β-synthase deficiency, tyrosinemia type I, or severe liver diseases were excluded from this study. No other positive results of blood physiological indicators for IEME were detected in MATD patients.MAT1A, GNMT, and AHCY) and Met-related IEMD, such as homocystinuria . Sanger sequencing was performed to verify variants detected in the panel. Novel missense variants were evaluated with several automatic tools . Variants leading to truncated protein were classified as pathogenic (P) or likely pathogenic (LP) according to the recommendations of the American College of Medical Genetics and Genomics (ACMG)  or by MyGenostics  during 2010–2015. Other samples were analyzed with a targeted gene panel by next-generation sequencing (NGS) in our Genetic Diagnostic Laboratory at the Children’s Hospital as previously described  and conts (ACMG) .MAT1A, the Met value and the ratio of Met/Phe from one parent carrying the same variant were detected.All positive cases were followed up by specialists at the Department of Genetics and Metabolism. Regular intervals ranged from every 1–3 months, depending on the severity of manifestations, and were followed for cases at 1 year and every 3–6 months for cases beyond 1 year. Data from profiles of serum amino acids (including methionine), tHcy, and routine blood tests for the functions of the liver and kidney were monitored regularly during the follow-up. Other indicators, such as blood gases and electrolytes, serum glucose, and lactic acid, were collected irregularly. Bayley Scales of Infant Development, or the Ages-Stages Questionnaire (ASQ) or Gesell Developmental Schedule were used to assess the developmental status of MATD patients. Length/height-for-age and weight-for-age standards were according to China growth standards (0–3 years old) and WHO Child Growth Standards (>3 years old). Some special patients with severe clinical abnormalities or with extreme elevation of plasma Met (>800 μmol/L) were referred to undergo a brain MRI (magnetic resonance imaging) scan. If suspected cases harbored an unreported variant of Interventions were considered when plasma Met was >500 µmol/Lin infants. A low-methionine formula is usually used. Vitamin B6 and betaine were considered when tHcy increased slightly twice or exceeded 30 μmol/L. One patient was given oral SAMe. One patient underwent liver transplantation.t-test or ordinary one-way ANOVA was performed for comparisons. p < .05 was considered significant.Met values were illustrated using GraphPad Prism 8.0. A 2-tail paired or unpaired Student’s p < .001, A total of 4,065,644 neonates were screened and 35 positive cases were diagnosed with MATD, which led to an estimated incidence of 1/116,161 in the MATD cohort of Zhejiang Province, a comparable incidence to that reported in Japanese . SimultaMAT1A, of which 11 individuals (P5-P15) carried c.791G>A/p. R264H and four individuals carried c.776C>T/p. A259V. Patient 1 (P1) and patient 9 (P9) were born prematurely, while their mothers’ pregnancies were uneventful to that point. Consistent with previous reports  diagnosed with a monoallelic variant of  reports , variant reports . All AD MAT1A without other pathogenic genes in this metabolic disorder. All patients were full-term infants. Compared to AD patients, AR patients have more complicated phenotypes and genotypes. The initial Met concentrations in 20 AR cases ranged from 60 to 332 μmol/L, with a mean value of 157 μmol/L , a negative result according to biochemical indicators in NBS.Twenty cases  of AR-type MATD were diagnosed with persistently high plasma Met concentrations and compound heterozygous or homozygous variants of 7 μmol/L . No sign7 μmol/L , which w7 μmol/L  and then7 μmol/L . All theAs MATD is a rare metabolic disorder with a limited population, detailed records of patients with neurological abnormalities are important evidence for better management of other cases in the future. Here, five patients with a Met value >800 μmol presented as follows: patients P16, P25, P27, and P31 had neurological abnormalities, and P35 carried 3 alleles with the same allele (c.695C>T/p. P232L) in P16.de novo variant c.695 C>T/p. P232L identified. Further evaluation of the intronic allele is needed. He had an initial Met 257 μmol/L, a high ratio of Met/Phe 4.58, and elevated plasma tHcy (59 μmol/L) with persistent hypermethioninaemia during follow-up. The recall Met was increased to 1283 μmol/L in the first month of life. Immediately, a diet with restricted protein and vitamin B6 was prescribed. At 2 months of age, the low-Met formula was added at a ratio of 5:1 to ordinary infant milk powder. The plasma Met levels during treatment were maintained at 403–599 μmol/L with a slight elevation of tHcy 17.6–19.0 μmol/L. At 20 months old, he had an intelligence score of 82 and an action score of 92 according to the Bailey Scale of Infant Development (BSID). Due to difficulties in persisting in a low-met restriction diet when grown, Met levels fluctuated within 1110–1165 μmol/L after 3 years of age. At age 7 years 9 m, brain MRI examination revealed a large range of abnormally high intensity in T2-weighted images with a reduced diffusion of white matter  values were observed with intermittent interventions. However, her development status was normal on the ASQ test, and both height and weight were P75 at follow-up at the age of 42 m.Patient 27 was homozygous for a reported variant c.769G>A/p. G257R with persistent hypermethioninaemia. During the first 3 months of life, his Met values were persistent at 1069, 1097, and 1408 μmol/L, respectively, with a low-Met formula diet. At the age of 10 m, he had a motor delay with a gross motor score of 10 and a fine motor score of five on the ASQ evaluation. After SAMe supplementation and rehabilitation training, his ASQ score climbed to normal at 48 m of age, with a gross motor score of 60 and a body weight of P50. However, MRI showed mild hyperintense T2-weighted images in the white matter of cerebral hemispheres and widened bilateral frontotemporal extracerebral space  at 4 yeaPatient 31 had compound heterozygous variants, c.769G>A/p. G257R and c.875G>T/p. R292L. With a Met-restricted diet, his Met was decreased from 977 μmol/L at age 1 m to 159 μmol/L at age 5 m and then manifested <500 mol/L. A transient development delay was observed at age 2 years, with a developmental quotient of 68 by the Gesell system, but disappeared at follow-up with a P50 score in both his height and weight at 5 years 10 m.Patient 35 harbored three alleles, a paternal c.695C>T/p. P232L variant and two novel maternal variants c.608T>A/p. I203N and c.922G>C/p. A308P, with an initial Met of 183 mol/L. his Met value was 600 μmol/L and then increased to 808 μmol/L at the age of 2 m. Given low Met intake and betaine treatment, the Met fluctuated between 543-595 μmol/L until the age of 13 m, with normal growth and development. However, his Met increased up to 713–982 μmol/L after the protein restriction diet was discontinuous. Both his height and weight were P75 as of follow-up at the age of 108 m.in silico. However, the novel truncating variants were postulated to be pathogenic (P) or likely pathogenic (LP) according to ACMG recommendations  were detected by the cutoff index in NBS, as shown in In this retrospective study, we described the phenotypes, genotypes, and long-term prognosis of 35 patients with MATD based on newborn screening, with an estimated incidence of 1:116,161, consistent with previous reports in Asia . NewbornNevertheless, the Met value is still the best primary marker for both diagnosis and prognosis. The initial value of the Met and Met/Phe ratio is not correlated with neurological abnormalities in this MATD cohort. However, a significant difference was found in the initial Met levels between patients and carriers. Similarly, significant differences were presented in mean plasma Met values during follow-up among AD-type patients (147 μmol/L), AR-type patients (479 μmol/L), and carriers (80 μmol/L). The high level of plasma Met is correlated with the severity of the disease to a certain extent. Patients with plasma Met values >800 μmol/L had neurological abnormalities as shown in P16 and P27 as elsewhere . AdditioMethionine restriction was most useful for managing plasma Met values in our patients as well as SAMe supplementation . The MetAR patients here had higher phenotypic and genetic heterogeneity than AD patients. The heterozygosity c.776C>T or c.791G>A surely caused no adverse long-range clinical effects on its patients . HoweverHere, our long-term prognostic data will be helpful for better understanding the natural history of MATD and optimal management for cases in the future. However, there were some limitations: 1) the detection of SAMe levels in the blood or cerebrospinal fluid (CSF) of patients was not performed; 2) whether the dose of the SAMe was dependent on weight or Met values was not clear; 3) limited by the small number of MATD patients and higher genetic heterogeneity, the relationship between phenotype and genotype is mostly undefined.


A multimodal antimicrobial stewardship intervention was associated with a decrease in antibiotic prescribing for targeted non–coronavirus disease 2019 (COVID-19) upper respiratory infections from 27.6% in 2019 to 7.6% in 2021. We describe our approach to prioritizing departments for 3 levels of interventions in the setting of limited stewardship personnel. As of 2020, institutions with ambulatory accreditation from The Joint Commission are required to implement an outpatient ASP under Standard MM.09.01.03. Components of this standard include the establishment of local leaders responsible for outpatient ASPs, selection of an annual goal related to antimicrobial use, adherence to evidence-based practices and guidelines pertaining to the annual goal, education of providers regarding ASP practices, and tracking and reporting of antimicrobial prescribing metrics.An estimated 30% of outpatient antibiotic prescriptions in the United States are likely inappropriate, which has prompted the need for antimicrobial stewardship programs (ASPs) in the outpatient setting. URIs, therefore, represent an optimal initial area of focus for new outpatient ASPs aiming to decrease inappropriate outpatient antibiotic prescribing.Acute upper respiratory infections (URIs) account for ∼44% of outpatient encounters in the United States that result in an antibiotic prescription, and only 50% of these prescriptions are likely appropriate.The purpose of this study was to describe the implementation of a multimodal ASP intervention prioritized by potential for department-level improvement and its impact on antibiotic prescribing for targeted non–coronavirus disease 2019 (COVID-19) URI encounters within a variety of outpatient settings in a community health system.The Mayo Clinic Health System–Southwest Minnesota comprises 5 hospitals with emergency departments and 13 outpatient clinics that include 3 urgent care departments, 2 pediatric and adolescent medicine departments, 1 community internal medicine department, and 11 family medicine departments. A single antimicrobial stewardship pharmacist is responsible for both inpatient and outpatient ASPs across the region. Diagnosis data were derived from International Classification of Disease, Tenth Revision (ICD-10) codes entered during visit-based encounters. URI ICD-10 codes were divided into diagnostic tiers using a previously validated schema based on whether antibiotics were always indicated (tier 1), sometimes indicated (tier 2), or not indicated (tier 3). The primary metric for the dashboard was antimicrobial prescribing rate for tier-3 diagnoses, calculated by dividing the total number of tier-3 visits in which antibiotics were prescribed by the total number of tier-3 visits for patients seen in visit-based encounters in the department specialties of family medicine, internal medicine, pediatrics, emergency medicine, and urgent care. ICD-10 codes for COVID-19 were not included in the denominator. Antimicrobial prescribing data can be viewed by region, clinic location, department specialty, and provider, and the data can be filtered by provider and patient demographics and by diagnosis.An outpatient ASP dashboard within the institutional electronic health record (EHR) was created using Epic SlicerDicer  to guide outpatient ASP efforts across the Mayo Clinic Enterprise.The outpatient ASP goal for the Mayo Clinic Enterprise in 2021 was to reduce antibiotic prescribing in tier-3 URI encounters, with each region being responsible for implementing local initiatives to achieve a specific regional goal. Resources created for use across the enterprise available by January 2021 included a slide set for provider education, a patient-facing commitment poster, an EHR order panel with syndrome-specific recommendations, and symptomatic management guides for patients.Mayo Clinic Health System–Southwest Minnesota set a goal to reduce antibiotic prescribing for tier-3 URI indications from 27.6% in 2019 to <21.6% in 2021. Baseline data from 2019–2020 were reviewed to prioritize local outpatient ASP initiatives. Two urgent-care departments with a large number of tier-3 encounters (>300 per year) and a high rate of antibiotic prescribing (>40%) were identified as high-priority targets. All family medicine and pediatric and adolescent medicine departments were identified as moderate-priority targets due to moderate-to-high baseline prescribing rates (15%–57%) across these specialties but a lower number of tier-3 URI encounters (∼100–300 per year). The remaining urgent-care department, all emergency medicine departments, and all community internal medicine departments were identified as low-priority targets due to low baseline prescribing (<15%) and/or low numbers of tier-3 encounters (<100 per year).At least 1 presentation was given to all departments between October 2020 and February 2021 to introduce the regional ASP goal and available resources. Communication techniques were discussed, and providers were encouraged to provide positive treatment recommendations for symptomatic management when not prescribing an antibiotic.In addition to receipt of departmental presentation(s), departments identified as moderate priority received quarterly data summaries in 2021 that included provider-specific antibiotic prescribing data (Fig. This project was performed as a quality initiative and did not require review by the institutional review board.Antibiotic prescribing during tier-3 URI encounters decreased from 27.6% in 2019 to 19.1% in 2020, with a further decrease to 7.6% in 2021 following the implementation of our intervention (Table  successfully implemented a similar multimodal URI intervention within a geriatric clinic. We were able to implement our initiative on a larger scale by prioritizing departments to receive different levels of intervention.We observed a decrease in antibiotic prescribing during outpatient tier-3 URI encounters in our community health system following implementation of a multimodal antimicrobial stewardship initiative. Chauhan et al We distributed provider-specific antibiotic prescribing data to moderate- and high-priority departments. Buehrle et al incorporated peer-comparison reporting within their multimodal outpatient ASP, and they observed a sustained decrease in antibiotic prescribing after peer-comparison reporting stopped. The creation of a provider-specific scoring tool for prioritizing ASP interventions directed toward individual providers may merit future investigation.We prioritized ASP interventions by department based on the volume of targeted encounters and baseline antibiotic prescribing rates. Previous studies have shown provider-level variability in antibiotic prescribing rates, supporting the use of peer-comparison data as an ASP strategy. noted a similar reduction in urgent-care antibiotic prescribing for non–COVID-19 respiratory conditions during the COVID-19 pandemic, particularly during telemedicine encounters. Although the reasons for this trend are unclear and likely multifactorial, this decrease in antibiotic prescribing is encouraging given prepandemic reports of high antibiotic prescribing rates during telemedicine URI encounters and associations with patient satisfaction scores.Antibiotic prescribing during targeted non–COVID-19 URI encounters began to decrease in 2020 prior to our ASP intervention. Ha et alThis study had several limitations. We used billing data to categorize URI encounters, and we did not evaluate the appropriateness of individual antibiotic prescriptions. We were not able to include prescriptions written outside encounter-based visits in our data model due to a lack required indications for antibiotic prescriptions in the outpatient setting. We did not apply statistical testing due to the retrospective, observational design of this study. Because we were not able to control for confounders such as the COVID-19 pandemic, our findings may be incidental and cannot be causally associated with the intervention.Implementation of a multimodal ASP intervention was associated with a decrease in antibiotic prescribing during targeted non–COVID-19 outpatient URI encounters. Prioritization of specific departments or providers for ASP intervention based on rate of antibiotic prescribing and total number of encounters of interest may be advantageous in settings with limited ASP personnel.


The 2011 guidelines for management of pediatric uncomplicated community acquired pneumonia (CAP) recommend the use of ampicillin or penicillin first line. We sought to evaluate improvement in adherence to these guidelines through antimicrobial stewardship interventions at a single institution to minimize unnecessary broad antimicrobials.A retrospective chart review was conducted of admitted patients aged 2 months - 21 years old with uncomplicated CAP. The pre-intervention phase was September 1, 2019-February 29, 2020 and the post-intervention phase included September 1-February 28 of 2021 and 2022. Antimicrobial stewardship interventions included the incorporation of clinical practice guidelines (CPG) into a new institutional CAP pathway (approved in June 2020) and subsequent education to ordering providers in the spring of 2020. Patients with complicated pneumonia or with comorbidities including sickle cell disease, chronic lung disease, neurologic conditions, congenital heart disease or patients who were immunocompromised were excluded. The prescribing patterns of specific physicians were recorded and adherence to CPG recommendations were assessed. The primary endpoint was to measure the reduction of broad spectrum antibiotics  to narrow spectrum antibiotics .p=0.0034) and for discharge antibiotics (p=0.0003)  between the pre- and post-intervention groups. Guideline adherence was 78.5%. No change in length of stay or treatment failure were observed.A total of 114 patients were included in the study; 72 pre-intervention and 42 post-intervention. Mean age was 5.4 years pre- and 6.5 years post-intervention. A significant reduction in broad spectrum antibiotic use was noted in the ED (p=< 0.0001), during the first 24 hours of admission (Antibiotic Prescriptions in the Emergency DepartmentAfter the implementation of the CPG guideline there was a 17% reduction in the number of times a combination of antibiotics were used, a 7.2% reduction of broad spectrum antibiotics and a 24% increase in the utilization of narrow spectrum antibiotics, p-value= <0.0001.Antibiotic Utilization 24 hours After Hospital AdmissionAfter the implementation of the CPG guideline there was a 20% reduction in the number of times a broad spectrum antibiotic was prescribed, p-value =0.0034.Discharge Antibiotic PrescriptionsAfter the implementation of the CPG guideline there was a 18% reduction of broad spectrum antibiotics and a 34% increase in the utilization of narrow spectrum antibiotics, p=0.0003.The CPG and educational interventions had a positive impact on the antibiotic management of children hospitalized, with an overall 4.7% reduction of broad spectrum antibiotics and a 28% increase in the utilization of narrow spectrum antibiotics. Continued education may improve CPG adherence.All Authors: No reported disclosures.


MET exon 14 skipping mutation (METex14m) is rare and occurs in approximately 1-4% of all non-small cell lung cancer (NSCLC) patients and approximately 2.8% of resected stage I-III NSCLC patients. Savolitinib is an oral, potent and highly selective type Ib MET inhibitor, which has been shown to be promising activity and acceptable safety profile in patients with advanced NSCLC harboring METex14m. Most recently, many studies have been probing into the feasibility and efficacy of target therapy for perioperative application in NSCLC. Interestingly, there are very few recorded cases of such treatments. Here, we presented that systemic treatment with the MET inhibitor savolitinib before surgery could provide the potential to prolong overall survival (OS) of patients with locally advanced potentially resectable NSCLC. A 49-year-old woman was diagnosed with stage IIIA (T2bN2M0) primary lung adenocarcinoma exhibiting a METex14m by real-time quantitative polymerase chain reaction (RT-qPCR). Given that the tumor load and the size of lymph nodes experienced a significant downstaging after the neoadjuvant treatment of savolitinib with 600mg once a day for 5 weeks, left lower lobectomy and systemic lymphadenectomy were successfully performed. The pathological response was 50% and the final postoperative pathological staging was pT1cN0M0, IA3 . The case provides empirical basis for the neoadjuvant treatment with savolitinib in METex14m-positive locally advanced primary lung adenocarcinoma, which will offer some innovative insights and clinical evidence for more effective clinical treatment of neoadjuvant targeted therapy for METex14m-positive NSCLC. MET proto-oncogene. MET is a novel therapeutic target for lung cancer and is closely related to the survival, prognosis and certain drug resistance of lung cancer patients (MET exon 14 skipping mutation (METex14m), MET kinase domain mutation, MET amplification and MET fusions are included in MET genomic alterations. METex14m is an independent oncogenic driver occurring in 1%-4% of non-small-cell lung cancer (NSCLC) patients. Patients with METex14m have a distinct clinicopathology and face-poor prognoses (The tyrosine-protein kinase Met (c-Met), also known as hepatocyte growth factor receptor (HGFR), is a heterodimer transmembrane tyrosine kinase receptor encoded by the patients . MET exorognoses .METex14m, such as savolitinib, capmatinib and tepotinib. Savolitinib is an oral, potent and highly selective type Ib MET inhibitor that has yielded promising activity and acceptable safety profile in patients with pulmonary sarcomatoid carcinoma and other NSCLCs harboring METex14m (ALK) fusion gene, epidermal growth factor receptor (EGFR) mutations, RET rearrangements and ROS proto-oncogene 1 (ROS1) rearrangements   . More imA 49-year-old female patient visited a local municipal tertiary hospital with symptoms including dry cough, chest tightness and voice hoarseness for 1 month. The contrast-enhanced chest CT scan on July 1, 2021 demonstrated a 3.8 cm × 2.9 cm abnormal lung mass in the lobe of left lung. Enhanced signal was not detected inside the mass. Left hilar and subcarinal lymphadenopathy were also observed  were absent. After active discussions with the multiple disciplinary team (MDT) including respiratory physicians, thoracic surgeons, and radiologists, we all agreed that potentially resectable stage IIIA primary adenocarcinoma after adjuvant therapy would improve prognosis, prolong survival, and improve quality of life. Considering the N2 lymph node metastases involvement, we offered neoadjuvant chemotherapy followed by surgical resection. However, the patient preferred neoadjuvant target therapy rather than neoadjuvant chemotherapy. Given that there was no neoadjuvant indication for MET inhibitors, after adequate communication with the patient and her family and receiving written informed consent, we advised the patient to receive the target drug savolitinib as a neoadjuvant therapy based on the results of genomic testing.After initial medical examination and assessment, the patient was admitted to our hospital on July 8, 2021. Stage IIIA (T2bN2M0) lung adenocarcinoma was confirmed on the basis of the 18F-FDG PET/CT results, CT guided lung puncture, and subsequent pathological diagnosis. Genetic tests  of the lung lesion biopsy revealed the results of th edition). The patient recovered well and the quality of life has been improved accordingly. For patients with completely resected IIIA-N2 stage NSCLC, adjuvant cisplatin-based chemotherapy for 4-6 cycles is recommended to prevent recurrence and improve survival by eradicating minimal residual disease (MRD). Therefore, the patient received following 5 cycles of pemetrexed plus carboplatin adjuvant chemotherapy. Up to now, the patient has been followed up postoperatively for 38 weeks with no sign of recurrence based on the last chest CT examination on June 3, 2022. The timeline for diagnosis and therapeutic interventions for the patient can be seen in On July 24, 2021, the patient received savolitinib, 600 mg orally, once daily as neoadjuvant therapy. Around three weeks later , the first evaluation of the therapeutic effect presented a partial response (PR) (target lesion shrank from 3.8 cm × 2.9 cm at baseline to 2.6 cm × 2.2 cm), on the basis of the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Additionally, the left hilar and subcarinal lymph node obviously shrank, and the short diameter did not exceed 7 mm Figure 3ALK-positive patients with pathologically confirmed N2 NSCLC, after received crizotinib at a starting dose of 250 mg twice daily, ten patients received an R0 resection and two patients achieved a pathological complete response to neoadjuvant crizotinib, which provided the evidence for neoadjuvant crizotinib in locally advanced NSCLC  NSCLC cannot be operated due to factors such as tumor size and/or location. The prognosis of patients with unresectable stage III NSCLC is poor even after concurrent chemoradiotherapy . In unreed NSCLC .EGFR and ALK inhibitors neoadjuvant targeted therapy in the disease control and pathological downstaging for early-stage NSCLC patients harboring the corresponding mutation, and some case reports also observed that target therapy provided effective radiologic and pathologic response in ALK, RET and ROS1-positive resectable NSCLC patients  has been the most common adverse effect of savolitinib during clinical trials and post-market surveillance  is ongoing (NCT04926831)  NSCLC with 4926831) . Given tMETex14m-positive locally advanced primary lung adenocarcinoma, which can provide a reference for clinical treatment of such patients. Neoadjuvant savolitinib targeted therapy in IIIA-N2 lung adenocarcinoma with METex14m could achieve pathological downstaging and increase the possibility of radical surgery and R0 resection. Additionally, monitoring liver function is necessary during savolitinib treatment. Along with an acceptable side effect, neoadjuvant targeted therapy probably deserves to be recommended for these patients with lung cancers that harbor a targetable oncogene, which may have much more impressive therapeutic effects than the platinum-based chemotherapy typically used for neoadjuvant therapy. The findings of this case would provide some inspiring insights for prospective clinical studies to further explore the clinical value of neoadjuvant targeted therapy for METex14m-positive NSCLC.Overall, the case presented primary clinical evidence that neoadjuvant savolitinib targeted therapy is an effective treatment for The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China  . The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.L-JC, J-QZ, and MF contributed to the conception and presentation of the case report. L-JC, J-QZ, and D-QX provided clinical expertise and interpretations. L-JC and J-QZ organized the MDT meeting. YF and WX recorded the detail of the MDT meeting. L-JC, J-QZ, H-LX, N-NH, Z-JL, MF, WX, and Z-MJ overall management and treat the patient. MF was responsible for the manuscript writing, literature review, and pictures production. C-MF contributed to manuscript revision, editing and proofreading. All authors contributed to the article and approved the submitted version.This work was supported by the Anhui Provincial Key Clinical Specialty Discipline Construction Program (2021szdzk05). The funding body had no role in the design of the study, and collection, analysis, and interpretation of data and writing the manuscript.We are thankful to Jing-Jing Kang from the Department of Endocrinology, Fuyang People’s Hospital, who provided of the baseline contrast-enhanced chest CT and PET-CT images of the case. The authors want to thank the colleagues in pathologic and radiologic department for providing the pictures in this case. Finally, we thank the patient for providing consent for this case report.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.


Baijiu-producing environment. Strain YHM-G was identified as Hyphopichia burtonii according to its morphological properties, physiological and biochemical characteristics, and ribosomal large subunit 26S rRNA gene D1/D2 domain sequence analysis. The optimal conditions for 3-Met production by YHM-G were obtained by single factor design, Plackett–Burman design, steepest ascent path design and response surface methodology as follows: 42.7 g/L glucose, pH 6, 0.9 g/L yeast extract, 6 g/L L-methionine (L-Met), culture temperature 28 °C, shaking speed 210 rpm, loading volume 50 mL/250 mL, inoculum size 0.5% (v/v), culturing period 48 h and 2.5 g/L Tween-80. Under these optimal conditions, the 3-Met production by strain YHM-G was 3.16 g/L, a value 88.1% higher than that before optimization. Strain YHM-G can also produce a variety of flavor compounds that are important for many foods. This strain thus has the potential to increase the abundance of 3-Met in some fermented foods and enhance their aroma profiles.3-Methylthio-1-propanol (3-Met) is widely used as a flavoring substance and an essential aroma ingredient in many foods. Producing 3-Met by microbial transformation is green and eco-friendly. In the present study, one strain, YHM-G, which produced a high level of 3-Met, was isolated from the  Baijiu, beer and wine and is naturally present in apples, tomatoes and other foods [3-Methylthio-1-propanol (3-Met), a sulfur-containing higher alcohol, is a crucial aroma component of many foods and beverages, such as soy sauce, cheese, er foods . It has At present, 3-Met is produced by chemical synthesis and biotechnological methods . With thSaccharomyces cerevisiae, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Zygosaccharomyces rouxi and Williopsis species [However, relatively few studies have reported on the ability of microorganisms to produce natural 3-Met, which mainly included a few types of yeast, such as  species ,7,8,9. T species ,8,10.With the demand for natural 3-Met increasing and the rapid development of genetic engineering technology, most studies have begun to use molecular biology techniques to modify genes to engineer strains with a high yield ,16,17. ABaijiu-making environment and to optimize its fermentation conditions. In this study, a yeast identified as Hyphopichia burtonii with a high yield of 3-Met was obtained from the Baijiu-brewing environment. Several statistical methods will be combined to optimize the fermentation conditions for this strain to further enhance its 3-Met production. To the best of the author’s knowledge, the strain discovered in this study is among the highest 3-Met yielding natural strains previously reported.Therefore, the objective of this study was to obtain a strain with a high 3-Met yield from the Daqu (a starter for Baijiu production) samples were collected from different Baijiu-making enterprises to screen yeast strains. 3-Met, L-Met and chromatographic grade methanol were purchased from Sigma-Aldrich . All other chemicals were analytical grade and commercially available unless otherwise stated. Yeast extract peptone dextrose (YPD) medium for screening and culturing strains, Wallerstein laboratory nutrient agar (WL) medium for strain morphology identification and Sorghum hydrolysate medium (SHM) for aroma compounds production were prepared as described previously [2PO4, 6 g K2HPO4, 2 g NaCl, 0.03 g ZnSO4, 0.01 g MgCl2 and 0.02 g FeCl2, pH 5.0. All media were autoclaved at 115 °C for 20 min.eviously . The iniDaqu sample was added to 9 mL sterilized ddH2O, then the mixture was diluted to 10−5, 10−6 and 10−7. After dilution, 0.1 mL suspensions with different concentrations were spread onto YPD plates and inverted cultured at 30 °C for 5 days. Single colonies were picked and streaked on YPD plates to obtain a pure culture. After being identified as a pure culture by microscopic examination, each isolated colony was stored at −80 °C. Each yeast strain was pre-cultured in YPD liquid medium, then 0.1 mL activated yeast cell solution, which was adjusted to a concentration of 1 × 106 cells/mL by using a blood cell counting plate, was inoculated into 50 mL fermentation medium and cultured in a 28 °C incubator at 180 rpm for 48 h to evaluate its ability to produce 3-Met. After fermentation, the cultures were centrifuged at 4 °C for 10 min at 13,751× g, the clear supernatants were filtered through a 0.22-µm filter and 3-Met production was analyzed by high performance liquid chromatography (HPLC). Each yeast strain was analyzed in parallel three time. The yeast that produced the largest amount of 3-Met was selected for further characterization.Yeasts were isolated as reported previously . One grahttps://www.ncbi.nlm.nih.gov/, accessed on 17 July 2022) with accession number ON999037 and a BLAST search was conducted against the Nucleotide collection (nr/nt) database using the BLASTN program. All reference sequences were from type or otherwise authenticated strains. Moreover, the overlapping fragment of D1/D2 domain of the ribosomal large subunit 26S rRNA gene was then aligned using ClustalW. The sequence alignment was filtered in ARB to exclude gaps, missing data, ambiguous nucleotide positions and lowercase positions. A phylogenetic tree was constructed by the neighbor-joining method (MEGA version 7 software) using the Kimura 2-parameter model [The target yeast strain YHM-G was identified by morphological and physiological characteristics and ribosomal large subunit 26S rRNA gene D1/D2 domain sequence, as described by Fan et al. . YHM-G wer model .w/w), NaCl concentration , ethanol concentration  and 3-Met concentration  by monitoring the optical density at 560 nm (OD560) of the YPD medium, as reported previously [The growth response was measured in a certain range of parameters, including temperature , pH (1–14), glucose concentration , temperature (X2), glucose concentration (X3), initial pH (X4), loading volume (X5), inoculum size (X6), the time point of L-Met addition (X7), culture time (X8), yeast extract concentration (X9), L-Met concentration (X10) and Tween-80 concentration (X11) were chosen for PB design to screen important variables for 3-Met production . The design with three factors (time point of L-Met addition (A), glucose concentration (B) and yeast extract concentration (C)) and three levels was listed in v/v) at 30 °C at a flow rate of 0.7 mL/min for 15 min. The UV-detector was used at a fixed wavelength of 215 nm. The chromatogram grade 3-Met  was prepared by adding 0, 1, 2, 3, 4 and 5 mg of the standard solutions (0–5 g/L) in 1.0 mL of ultrapure water into a serial concentration to set up the calibration curve. The standard calibration curve working solution was analyzed by HPLC. The calibration curve was obtained and used to calculate of 3-Met in the sample. The analysis was performed in triplicate. The aroma compounds were analyzed by headspace solid–phase microextraction gas chromatography–mass spectrometry (HS–SPME–GC–MS) with a TraceGC TSQ 8000 Evo instrument  according to our previous report [m/z 33–550. The splitless mode was conducted for these analyses. The aroma compounds that the mass spectra with positive and negative matches higher than 800 with the National Institute of Standards and Technology library are reported. The amount of each aroma compound was calculated as the ratio of the mass concentration of tetraoctanol to the mass concentration of the compound.3-Met concentration were determined using a 1260 series HPLC  equipped with a ZORBAX Eclipse Plus C18 column  and a UV detector . After tp < 0.05) with Tukey’s test. Assays have been conducted at least in triplicate, and the reported values correspond to the mean value and standard deviation. SPSS 24.0 , OriginPro 9.1 , Design-expert 11  and Excel 2019  were used to process and analyze data.Statistical differences in the assessed strategies were analyzed using a one-way ANOVA . According to branch clustering, strain YHM-G was closest to H. burtonii CBS:2352 (KY107882.1) and H. burtonii CBS:232.37 (MH867400.1) in the phylogenetic tree constructed using MEGA 7.0.14 software  based on the neighbor-joining statistical method , thus strain YHM-G can be considered a yeast with high ethanol tolerance [S. cerevisiae CEN.PK113-7D, indicating that strain YHM-G has the potential to produce more 3-Met [Baijiu. Therefore, strain YHM-G could be used for brewing these foods and increasing their contents of 3-Met.3-Met not only has outstanding flavor characteristics, which endow specific flavor characteristics to traditionally brewed food products, such as activity ,37. Thernd 3-Met . The optnd 3-Met a, which  process ,40,41,42nd pH 12 a, a rangnd pH 12 a. This r process ,39,40. Ae YX3307 ,44,45,46e YX3307 . Comparee YX3307 ,48. Howee YX3307 ,50. As te YX3307 ,51. The olerance . The abire 3-Met . OverallAlthough producing 3-Met by yeasts from sugars via de novo biosynthesis has been rare, previous reports have emphasized that its biosynthesis by yeasts requires sugars ,3. ThereYeast extracts contain many vitamins and microelements, which can be used for yeast growth and affect the accumulation of yeast metabolites ,43,56. IAs L-Met is the precursor for 3-Met synthesis in the Ehrlich pathway, its concentration significantly influences 3-Met synthesis by yeasts ,59. LikeAlthough our results have confirmed the importance of L-Met for 3-Met synthesis by strain YHM-G, the question arises as to when adding L-Met could promote 3-Met synthesis. At present, no information detailing the optimal time for L-Met addition has been reported. The optimal time at which L-Met should be added for 3-Met production by strain YHM-G was determined by adding it at different culture times. 3-Met production was significantly higher when L-Met was added after 24 h of culture compared with adding at the start of culture, with no significant increase in production at 12 or 36 h but lower production at other times c. The reBaijiu making [Studies have shown that fluctuations in the environmental pH can stress microorganisms, thus changing the permeability of cell membranes and the activity of enzymes. The pH can change the state of nutrients in the system and affect the utilization of nutrients by microorganisms, resulting in different growth states and products for the microorganisms ,25,62,63K. lactis KL71 [Temperature is an important factor affecting the growth of microorganisms and the production of metabolites. If the temperature is too low, microbial growth will be greatly compromised; an excessive temperature may lead to cell death or enzyme inactivation, discouraging the accumulation of the end-products ,64,65. Btis KL71 , which wtis KL71 ,5. At thtis KL71 . A reductis KL71 ,66.Although yeasts are facultative anaerobic microorganisms, their growth and most biological metabolic activities benefit from aerobic conditions ,43. The 6 cells/mL by using a blood cell counting plate), can lead to low 3-Met production owing to the fact that longer delay period may lead to lower biomass, whereas a high inoculum volume, such as 6.4%, would also decrease the yield because excessive cell growth may lead to insufficient oxygen supply . The coefficient of determination (R2) was 0.9998, suggesting the model could be used to guide further study. Based on the Pareto chart  using Fisher’s test, the model was significant as the to chart , all eleficients . Thus, aBased on the above regression analysis of PB design, a design matrix of six runs with eleven factors, with their step lengths as the steepest ascent method and experimental experience, according to Equation E1, were used as the steepest ascent path design . The resAccording to the results of PB and the steepest ascent path design, a three-factor  and three-level BBD were used to optimize the best fermentation conditions for 3-Met production by YHM-G. Various maximum and minimum levels of three factors were used for the 15 experimental runs in A second order polynomial equation E2 was obtained to explain 3-Met production by applying multiple regression analysis on the experimental data:F-test) shows that the model was statistically significant. The R2 was 0.9709, indicating that the sample variation of 97.09% for 3-Met production was attributed to the independent variables and only about 2.93% of the total variation was unexplained by the model , and C (yeast extract concentration) had a significant positive effect on 3-Met production  response surface plots for 3-Met production described by the above model were drawn to illustrate the effects of the independent variables and to depict the interactions between two variables by keeping the third variables at their zero levels . As showv/v), time point of L-Met addition 4.9 h, culturing period 48 h and 2.5 g/L Tween-80. Validation experiments were performed at the optimal level, and the yield of 3-Met was 3.16 g/L, which was 88.1% higher than the value before optimization  = 4.9 h, B (glucose concentration) = 42.7 g/L and C (yeast extract concentration) = 0.9 g/L. According to the results of the statistically designed experiments, the optimized process parameters were: 42.7 g/L glucose, pH 6, 0.9 g/L yeast extract, 6 g/L L-Met, 28 °C, shaking speed 210 rpm, loading volume 50 mL/250 mL, inoculum size 0.5% (Baijiu-making environment. The analysis of its flavor-producing characteristics would encourage its application in fermented foods. The results showed that a total of 23 flavor substances, including seven alcohols, five phenols, three esters, two alkanes, one ketone, one aldehyde, one olefin, one furan, one sulfur-containing compound and one nitrogen-containing compound, were detected in SHM after culturing strain YHM-G for 48 h (Flavor substances affect food quality to a certain extent, with the unique flavor characteristics of fermented foods being mainly transformed from the ingredients in the substrate by microorganisms . Thus, mfor 48 h . Comparefor 48 h b. This oBaijiu-producing environment. Of these, one strain of yeast, YHM-G, produced the most 3-Met. This strain was later identified as H. burtonii, which had not been previously reported to produce 3-Met. The strain exhibited high tolerance to sodium chloride, glucose and 3-Met. The yield of 3-Met was 3.16 g/L, which, after optimization by single factor design, PB design, steepest ascent path design and RSM design, was 1.88 times higher than that of the original culture used. The strain YHM-G can also produce many other flavor compounds, such as alcohols, phenols and esters, which are considered important for assuring the quality of many traditional foods. Thus, the H. burtonii strain YHM-G has potential applications for improving the quality of some traditional foods.In this study, four strains of yeast that produced a high yield of 3-Met were isolated from the