The IZO thin-film transistor (TFT) prepared with femtosecond laser pre-annealing with low pulse energy generation (power of 3 W at 700 nm wavelength) for 100 s has also exhibited significantly improved electrical performance, the saturation mobility increased to 4.91 cm2/Vs, the Ion/Ioff ratio was enhanced from 4.5 × 105 to 2.1 × 106, the threshold voltage improved from − 1.44 to − 0.25 V, and the subthreshold swing was reduced from 1.21 to 0.61 V/dec. In conclusion, IZO TFTs with improved performance can be prepared using a femtosecond laser pre-annealing process, which has great potential for fabricating low-cost, high-performance devices.This paper examined the effects of no treatment versus plasma treatment, and femtosecond laser irradiation as pre-annealing processes on indium zinc oxide (IZO) films and annealing at high temperatures. The plasma pre-annealed multilayer stacked IZO TFTs showed better electrical properties with mobility enhancement from 2.45 to 7.81 cm Indium oxide, indium zinc oxide (IZO), indium gallium zinc oxide, and several other metal oxides have been utilized10. Multi-stacked IZO layers have lower porosity and higher density than single layers and can generate more electron carriers and exhibit improved electrical properties. Multi-stacked IZO films need to be annealed several times during preparation to obtain high-density films12. Multi-stacked IZO TFTs can be fabricated using solution processes, vacuum evaporation, chemical vapor deposition, and atomic layer deposition techniques. Of these, solution processes have the advantages of simple fabrication, low-temperature processing, and low cost19. In addition, oxygen (O2) plasma treatment, photochemical activation, femtosecond laser treatment, and thermal annealing under an O2 atmosphere were shown to improve the surface quality of the films and enhance the electrical properties of multi-stacked IZO TFTs22. The plasma pre-annealing treatment at low power (< 150 W) was demonstrated to improve the electrical properties of oxide TFTs by reducing the charge carrier concentration and conductivity, and offers the advantages of low cost, high efficiency, and large area uniformity24. A pre-annealing process using femtosecond lasers applies the laser to a specific spot of the film, resulting in less thermal damage in the film through which the laser passes. Femtosecond laser technology is used widely for TFT preparation because of its relatively short laser pulses and high transient intensity, which reduces the cost and avoids damage to oxide films26.IZO is one of the widely used active layer materials for TFTs because of its wide bandgap (3.68–3.76 eV), high mobility, high transparency, and smooth surfaceIn this study, TFTs composed of IZO films treated with different pre-annealing processes of plasma and femtosecond laser were fabricated to achieve high mobility and relatively good electrical stability and provide a relatively simple, low-cost preparation method. In addition, the effects of different pre-annealing processes on IZO TFTs were compared.A multi-stacked IZO film was manufactured on the a heavily dopped n-type Silicon bottom gate substrate that has a hot-grown 100 nm thick Silicon dioxide layer used a dielectric layer, as shown in Fig. 3)3·xH2O] in 2-methoxyethanol was prepared in a glass vial, and 50 μl of acetylacetone, and 22.5 μl of ammonia were added. Subsequently, 1.5 ml of a 0.1 M zinc acetate dihydrate [Zn(CH3COO)2·2H2O] solution in 2-methoxyethanol was prepared, and 30 μl acetylacetone was added. Both solutions were placed in a magnetic stirrer at 700 rpm and stirred at 60 °C for 1 h. The two solutions were mixed at a 1:1 ratio and stirred at 500 rpm and room temperature for 2 h to prepare the IZO precursor solution. The mixed solution was filtered through a 0.2 μm syringe filter to remove particulates and to obtain a more transparent and homogeneous solution, which is beneficial for spin-coating. The precursor solution was spin-coated on four clean silicon substrates at 1,500 rpm for 30 s, followed by a 30 min annealing process at 400 °C. This resulted in an IZO thickness of 20 nm. The same spin-coating and annealing processes were repeated to form the second layer. For forming the third layer, the spin-coating process under the same conditions was repeated again, but with different pre-annealing conditions. To fabricate the devices with plasma treated layers, the IZO layer was treated with oxygen plasma at an RF power level of 150 W for 3 min  generated at low pulse energy with a power of 3 W and a 700 nm wavelength was used for 100 s. The schematics of the effect of pre-annealing is shown in Fig. 2.5 ml of a 0.1 M precursor solution of indium nitrate hydrate [In and X-ray photoelectron spectroscopy (XPS). The electrical characteristics of the output, transfer, and stability of the TFTs were measured using a semiconductor parameter analyzer  in a dark room at room temperature.28. This suggests that a proper pre-annealing process involving plasma or femtosecond laser treatments can lead to more homogeneous multi-stacked IZO films.The surface morphology of the multi-stacked IZO films treated with different pre-annealing methods was analyzed by AFM; Fig. ds) at constant gate bias voltages (Vgs) of 0, 10, 20, and 30 V, drain bias voltage (Vds) incremented from 0 to 30 V in 10 V increments to investigate the electrical characteristics of the trhee types of IZO TFTs. All devices exhibited typical n-type output characteristics, but the one with femtosecond laser and plasma treated IZO had smoother and more stable saturation curves than the other IZO TFTs. On the other hand, the femtosecond laser and plasma treated IZO TFTs showed leakage currents at low drain voltages. Figure ds fixed at 30 V as Vgs was increased from − 10 V to 30 V in 0.5 V increments. All the TFTs showed typical n-type characteristics as Ids increased with Vgs and exhibited a significant transition from the off-state to the on-state.Figure sat), threshold voltage (Vth), on/off current ratios (Ion/Ioff), and subthreshold swing (SS) values of the pristine, plasma, and femtosecond laser pre-annealed multi-stacked IZO TFTs. Compared to the device with pristine IZO, which had a mobility of μsat = 2.45 cm2/Vs, the devices with plasma treatment had higher electron mobility (μsat = 7.81 cm2/Vs).The ones with the femtosecond laser treatment also showed improved performance (μsat = 4.91 cm2/Vs). On the other hand, the Ion/Ioff of the plasma-treated IZO TFT decreased compared to the pristine one, with Vth shifting from − 1.44 V to 1.13 V in the positive direction. By contrast, the femtosecond laser treatment resulted in an increase in Ion/Ioff to 2.1 × 106, and an improved Vth shift to − 0.25 V. Significant improvements in the SS values were also observed for both pre-annealed IZO TFTs, particularly for the femtosecond laser-treated ones, from the 1.21 V/dec of the pristine device to 0.61 V/dec. The improved electrical properties of the devices correlated with the lower SS values, which relate to the interfacial trap density (Nit), as expressed in Eq. , respectively , as shown in Fig. th caused by charge trapping31. The pristine IZO surface exhibited the highest attenuation of the drain current due to the presence of many defects, with the normalized Ids dropping to a final value of 0.77 after 600 s. The normalized Ids of the plasma treated devices has dropped to 0.69, and for the femtosecond laser one it decreased to 0.77 during the same time interval. The femtosecond laser irradiated devices show improved stability. On the long term, the device may deteriorate due to environmental factors such as water, oxygen, etc. Measuring the device performance 10 days after the fabrication, for the pristine device, a tenfold increase in the off curent was observed, but other parts of the transfer curve has remained relatively unchanged, as shown in Fig. The bias stress stability characteristics of the pristine, plasma, and femtosecond laser-treated TFTs were measured. The time evolution of I1), 531.2 (O2), and 532.4 (O3) eV, which were assigned to metal-bonded oxygen (M–O), oxygen vacancies (OV), and hydroxide species (O–H), respectively. Figure V peak area, but the area of M–O peak decreased. OV is generally considered to be a shallow donor that is formed by the breakage of In–O and Zn–O bonds. Compared to the pristine IZO TFTs, the oxygen atmosphere in plasma pre-annealing promoted the decomposition of M–O, increasing the OV and improving the carrier concentration, and affecting the associated mobility. The relative area of the M–O lattice peaks of the femtosecond laser pre-annealed films were reduced relative to the original, and the area of the OV peaks was increased32. The variation of the electrical properties is consistent with the trends in Table The effect of oxygen content of the IZO films on the electrical properties of the devices was studied. The multilayer structured IZO films were examined by XPS to determine the effects of pre-annealing methods of plasma and femtosecond laser treatment on the IZO films compared to the pristine sample device, as shown in Fig. 2/Vs. The optimized preparation process for the femtosecond laser pre-annealed IZO TFTs was demonstrated, showing an improved Ion/Ioff enhancement to 2.1 × 106. Femtosecond laser pre-annealing has important theoretical and practical implications for improving the electrical properties and time stress stability of oxide IZO TFTs for the low-cost fabrication of amorphous oxide electronics.The effects of plasma, and femtosecond laser pre-annealing processes on the performance of multi-stacked IZO-based devices were studied by comparing the characteristics to devices with pristine multi-stack IZO active layers. The plasma pre-annealed multi-stacked IZO TFTs showed improved electrical properties with a mobility of 7.81 cm


Abnormal activation of the β-catenin signaling pathway is involved in various malignancies, including breast carcinoma.Aberrant expression of β-catenin has been associated with more aggressive behaviors of breast cancer in some previous studies. . In the present study, we intend to evaluate the β-catenin expression in breast cancer specimens and study its relationship with clinicopathological parameters. In this cross-sectional study,88 samples diagnosed as invasive ductal breast carcinoma from 2007 to 2017 were evaluated. The slides and paraffin blocks were retrieved from the archive of pathology department. Patients’ clinical characteristics and other information were also extracted from medical documents. Sections from related paraffin blocks through the tissue microarray method were provided, and immunohistochemistry staining for β-catenin was done. Then different patterns of β-catenin expression and the relationship between different patterns and clinicopathological parameters were investigated.Of the 88 breast cancer samples, 94% were female, and 6% were male. In 70% of the samples, normal membrane expression of β-catenin was observed. Whereas in 30% of them, aberrant expression of β-catenin was observed. A close significant relationship was observed between aberrant β-catenin expression and age over 50 years  and negative HER2 . In the present study, a correlation was observed between aberrant β-catenin expression and age over 50 years in patients and HER2 negativity, although this association was not statistically significant. Breast cancer is the most common malignancy in women worldwide and is the second leading cause of cancer death . Current treatments for breast cancer depend on estrogen receptor(ER), Progesterone receptor (PR), and Human epidermal growth factor receptor2 (HER2) status .Knowledge of clinicopathological parameters such as tumor size, tumor grade, and lymph node involvement status is essential in clinical decision making for the patient .In addition to the above, identifying novel biomarkers involved in tumor invasion and metastasis can help determine the tumor’s prognosis and new targeted therapies . Several signaling pathways, including Wnt, are involved in breast cancer . β-catenin is an oncogene  and is an essential intermediate in the Wnt signaling pathway. E-cadherin is a molecule involved in cell-cell adhesion in the plasma membrane and helps maintain cell polarity . It has an intracellular region that contains the binding site to interact with catenins .Some studies have suggested the role of β-catenin as a proto-oncogene, which causes many cancers, such as colon cancer .Recent studies have shown that Wnt/ β-catenin activation and abnormal cytoplasmic/nuclear expression of β-catenin in many human cancers, including breast carcinoma, are associated with increased invasion, metastasis, and poor prognosis .It has also been observed that nuclear and cytoplasmic accumulation of β-catenin, which represents Wnt pathway activation, is more common in basal-like breast cancers, and a significant correlation was found between the aberrant expression of this maker and negativity of hormone receptors and HER2. Due to the lack of sufficient information in this area, particularly in Iran, in the present study, we examined the expression and location of β-catenin in breast cancer specimens and its relationship with clinicopathological parameters.In this cross-sectional study,88 samples diagnosed as invasive ductal breast carcinoma who underwent mastectomy from 2007 to 2017 were evaluated. The slides and paraffin blocks were retrieved from the archive of the pathology department of Shariati and Sina hospitals, Tehran university of medical science. Tumoral tissue with representative areas  were identified and included. The tumor areas selected from the H&E slide were matched to the corresponding block and used for tissue microarray construction. Selected 0.6 mm tissue cores from the donor block were placed in a recipient block, and 5-micron sections were cut from the recipient block and then transferred to a slide. For increasing the accuracy, two cores from each tumor were placed on the slide, and the expression of β-catenin marker through immunohistochemical staining was examined. IHC was performed using Rabbit anti-human β-catenin monoclonal antibody  according to the manufacturer’s protocol. Two pathologists re-examined tumoral tissue from each patient, and different patterns of β-catenin expression were examined. Patients’ clinical characteristics, including age, sex, and other information include tumor size, probable lymph node involvement, hormonal profile, and expression of Her2 and Ki67 IHC markers, were investigated. These data were also extracted from medical documents, and the relationship between β-catenin expression and clinicopathological parameters was investigated.Representative H&E slides were examined and graded according to Nottingham modification of the scarf-Bloom-Richardson (MBR). The results related to qualitative variables were calculated and reported as frequency and percentage, and the results about quantitative variables were calculated as average and standard deviation.Chi-squared test and Fisher’s Exact test were used to evaluate the relationships between qualitative variables. After data collection, the analysis of data was conducted with SPSS version 22.0 software. P-value <0.05 was considered significant.In this study, the researchers did not intervene, and no cost was imposed on the patient. The results of the survey were reported without mentioning the names and details of the patients. As a result, this study is morally acceptable.In total, 88 histologically confirmed invasive breast cancer cases were included in this study and analyzed by IHC for β-catenin expression. Of these, 83(94%) were female, and 5 (6%) were male. The patients’ mean average age was 52 +_13.8 years ( ranged from 27-91 years).The frequency of different patterns of β-catenin expression is shown in There was no significant correlation between gender and aberrant β-catenin expression.. Also, there was no significant correlation between mean age and aberrant β-catenin expression. However, aberrant expression of β-catenin appears to be more common in people over 50 years of age.The relationship between HER2 and aberrant expression of β-catenin is shown in β-catenin is involved in carcinogenesis Through the Wnt signaling pathway. This pathway’s activation causes nuclear and cytoplasmic expression of this marker . Under normal conditions, this marker is expressed in the epithelial cell’s cytoplasmic membrane .Aberrant activation of the β-catenin signaling pathway has been observed in many malignancies, including breast cancer. However, its importance in breast cancer is still not well understood.Contradictory results have been reported regarding the relationship between β-catenin expression and clinicopathological variables. Therefore, there is a need for extensive research in this area .In breast cancer, the type of treatment depends on the hormone receptors, Her2, and the clinical-pathological variables, including tumor size, tumor grade, and lymph node involvement status . Therefore, recognizing new biomarkers can be a step towards identifying new treatments for this common cancer.Previous studies have shown that β-catenin signaling activation in the Wnt pathway and aberrant expression of this marker is associated with tumor progression and metastasis . Therefore, familiarity with this biomarker is probably useful in determining patients’ prognosis and survival and recognizing new treatments.Due to the high prevalence of breast cancer and the lack of sufficient information in this area, particularly in Iran9, in the present study, we examined the expression and location of β-catenin in breast cancer specimens and its relationship with clinicopathological parameters.In our study, the Tissue microarray (TMA) method was used. In this method, hundreds of tissue samples can be examined simultaneously under the same conditions. Therefore, it saves time, money, and consumption of the reagent. Also, the amount of tissue required in this method is low, and tissues can be preserved for further studies . After the TMA blocks described in the material and method section were prepared, They underwent immunohistochemical staining for the β-catenin, and different staining patterns of this marker were examined.In our study, about 70% of the samples showed normal β-catenin membrane expression. In about 30% of the samples, the aberrant expression of this marker was observed. As previously mentioned, aberrant nuclear and cytoplasmic expression of this marker is associated with invasion, metastasis, and poor prognosis .In a study, Varma (2020) examined the association of different β-catenin expression patterns with breast carcinoma pathogenesis . In this study, the expression of β-catenin and Cyclin D1 was evaluated by immunohistochemistry in 82 breast cancer cases. Abnormal β-catenin expression was seen in 80% of breast invasive ductal carcinoma cases.In the study of wang et al., (2015) The clinical implications of β-catenin protein expression in breast cancer were investigated. In this study, out of 241 patients with breast cancer who underwent radical surgery, abnormal β-catenin expression was observed in 41 patients.In a study, Lee (2005) examined the prognostic significance of abnormal β-catenin expression in breast carcinoma. In this study, abnormal β-catenin expression was observed in 30 of 55 breast carcinoma cases.In the study of Shen et al., (2016) The prognostic value of β-catenin and E-cadherin in triple-negative breast cancers was investigated. Most cases of triple-negative breast cancers had an abnormal β-catenin expression.In a study conducted by Geyer et al., (2011) abnormal β-catenin expression was significantly associated with ER and HER2 negativity. While in the study of Niu et al., (2009) There was a significant relationship between HER2 positivity and abnormal β-catenin expression.Our study investigates the relationship between HER2 and β-catenin expression in patients. Once the Her2 variable was considered a ranking variable, There was no significant correlation between Her2 expression and β-catenin protein expression using the Chi-square test. Considering Her2 as a two-state variable (negative and positive), this relationship is meaningful at the 10% level. In the study of Niu et al., (2009), abnormal β-catenin expression was associated with positive lymph node status and higher histological grade.In our study, the median Ki67 percentage was 15%. Tumors were divided into two groups based on the Ki67 rate ( ≤15% and >15%). In our study, there was no significant relationship between Ki67 expression and abnormal β-catenin expression. Also, there was no meaningful relationship between patient sex, histological tumor grade, and lymph node metastasis with abnormal β-catenin expression.In the study of Shen et al., (2016) Tumor size, pathologic tumor stage, lymph node involvement, and decreased β-catenin membrane expression were associated with poor prognosis and decreased patient survival.In our study, the tumor’s median size was 4 cm, and the tumors were divided into two groups in terms of size (≤ 4cm and >4cm). There was no significant relationship between tumor size and abnormal expression of β-catenin.In a study by Lee (2005) abnormal β-catenin expression was significantly associated with lymph node metastasis and poor prognosis in patients.In a Study by Khramtsov et al., (2010) abnormal nuclear and cytoplasmic β-catenin expression was higher in basal-like breast cancers than in others. Also, in this study, the survival rate in patients was evaluated, Which was found in cases where there was an abnormal expression of β-catenin, survival was lower than β-catenin membranous expression.In the study of Khan et al., (2018) deregulation of the Wnt pathway was not associated with the age of onset of the disease, the tumor grade, and triple-negative breast cancers.Our study observed a correlation between aberrant β-catenin expression and age over 50 years in patients, although this association was not statistically significant.In a study by Lee (2005) β-catenin lack of membrane staining was associated with a higher grade of tumor and negative ER. There was no significant relationship between ER and PR positivity and the β-catenin’s abnormal expression in our study. One of the critical limitations of our study is the lack of clinical outcomes in patients. This limitation was due to the lack of access to patients’ contact information. Besides, information about the patients’ clinical-stage was not available.In conclusion, based on previous studies’ results, an aberrant β-catenin expression is associated with the tumor’s aggressive behavior, metastasis, and poor prognosis in breast carcinomas. As discussed, there are conflicting results in various studies regarding aberrant β-catenin expression with clinicopathological parameters. In our study, a correlation was observed between aberrant β-catenin expression and age over 50 years in patients and HER2 negativity, although this association was not statistically significant.What is clear from the results of some studies is that β-catenin can be used as a prognostic marker and predictor of survival in patients. Therefore, familiarity and use of this marker can help determine patients’ survival and discover new breast cancer treatments. We propose further studies with larger sample sizes and information from patient follow-up.None.


Drosophila. Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad  to control neuromuscular junction (NMJ) growth in  Our study contributes to a broader understanding of AP2 complex-dependent regulation of synaptic growth signaling, which might have implications in neurodevelopment under conditions where endocytosis and trafficking of the BMP receptors are perturbed.Understanding the regulation of synaptic development and refinement could provide mechanistic insights into the neural basis of fundamental processes such as learning, memory and adaptation. In this study, we describe the role of σDrosophila neuromuscular junction (NMJ) synapses is a well-studied growth-promoting pathway (Drosophila) and its translocation into the motor neuron nuclei, followed by the transcription of BMP target genes , Wingless (Wnt), and Ubiquitin-Proteasome-mediated protein degradation  pathways . Besides pathway . The canet genes .Drosophila NMJ, the retrograde BMP signaling is initiated by secretion of Glass bottom boat (Gbb) from the muscle and the motor neurons  and aberrant synaptic growth. For instance, it is not known whether specific modes of endocytosis externalize specific cargos whose trafficking defect perturbs synaptic signaling. CME, the major endocytic pathway, is required not only for basal synaptic transmission at nerve terminals but also for synapse development . For insosophila . It is uosophila .In central synapses, AP2-dependent CME is dispensable for membrane regeneration from the presynaptic plasma membrane following high-frequency nerve stimulation . Howeverσ2-adaptin mutants : 7tkv (BL3242), 13Med (BL7340), A12wit (BL5173), j1E4Dad (BL10305), KG02457AP2σ (BL13478), UAS-Rab11-GFP (BL8506), k00237Mad (BL10474), α-adaptin dsRNA (BL32866), Clc dsRNA (BL27496), 1Synj (BL24883), 2Synj (BL24884). Other lines used in this study are: D42-Gal4  to expose the NMJs and fixed in 4% paraformaldehyde in PBS (pH 7.2) for 30 min at room temperature. Fillets were then washed in PBS containing 0.15% Triton X-100, blocked for 1 h with 5% bovine serum albumin (BSA), followed by overnight incubation with primary antibody at 4°C. The monoclonal antibody anti-CSP (1:100) was obtained from the Developmental Studies Hybridoma Bank (DSHB). The polyclonal antibody against Rab5  were recorded for 60 s, followed by recordings of EJPs at 1 Hz stimulation. For High-frequency recording, nerves were stimulated at 10 Hz, and EJPs were recorded for 5 min. For recording EJPs, stimulation pulse was delivered using Grass S88 stimulator . The signals were amplified using Axoclamp 900A, digitized using Digidata 1440A, and acquired using pClamp10 software (Molecular Devices). Muscles with resting membrane potential between −60 and −75 mV were used for analysis. The data were analyzed using the Mini Analysis program .All intracellular recordings were performed on wandering third instar larvae as described previously . BrieflyConfocal images of muscle four NMJ at A2 hemisegment were used to plot the intensity profile. A single bouton section was used, and the intensities of Tkv and HRP were analyzed by drawing a line across the bouton using Fiji/ImageJ software. The graph was plotted in Microsoft Excel using the intensity values obtained from Fiji/ImageJ software. As the intensity of Tkv in control was too low to plot the graph, all the intensity values were multiplied two-fold.m cacodylate buffer containing 2% glutaraldehyde for 10 min at room temperature, transferred to a fresh fixative, and kept overnight at 4°C. The fillets were postfixed for 1 h with 2% osmium tetroxide (OsO4) solution prepared in 0.12 m cacodylate buffer. The samples were rinsed with 0.12 m cacodylate buffer followed by washes with distilled water to avoid precipitation of cacodylate with Uranyl acetate. Subsequently, the samples were subjected to en bloc staining with 2% uranyl acetate. The stained fillets were again washed with distilled water and dehydrated using graded solutions of ethanol before final infiltration of the samples through propylene oxide for 30 min. Stained and dehydrated fillets were embedded in epoxy resin and hardened overnight at 60°C. Muscles embedded in epoxy resin were sectioned at 60 nm. Ultrathin sections of the muscles stained with 2% uranyl acetate (in 70% ethanol) and 1% aqueous lead citrate were examined at 120 KV on a Tecnai G2 Spirit BioTWIN (FEI) electron microscope. The number of synaptic vesicles per bouton were counted manually using the Multi-point tool in ImageJ/Fiji software and then divided by their respective bouton areas to obtain the vesicle density/μm2 area of a bouton. For vesicle size, diameters of at least 100 vesicles from 10 bouton sections of each genotype were used for quantification. For calculating the number of synaptic vesicles docked at the active zones, only those vesicles that were touching the T-bar platform were counted. For calculating the SSR thickness, the scale bar in the images was first calibrated to the number of pixels using the Set scale function in ImageJ/Fiji software. This was followed by using the Straight-line tool to draw a line across the SSR and Measure tool to calculate the thickness.TEM was performed as described previously . Brieflym Tris-HCl, pH 6.8, 25 mm KCl, 2 mm EDTA, 0.3 m sucrose, and 2% SDS in water. The homogenized sample was then mixed with an equal volume of 2× Laemmli buffer. The protein equivalent to 50 μg was separated on 12% SDS-PAGE and transferred to Hybond-LFP PVDF membrane . The membrane was then blocked with 5% skimmed milk for 1 h, followed by overnight incubation with anti-Rab11 (1:2000) and anti-Tubulin (1:5000) antibody. IRDye 800 (1:10 000) was used as a secondary antibody, and signals were visualized on LI-COR Odyssey platform. The density of Western bands was quantified using ImageJ/Fiji software.The western blot analysis was done as previously described previouusly . Brieflypost hoc Tukey’s test, or Student’s t test was used. GraphPad Prism 8 was used to plot the graph. Error bars in all the histograms represent SEM. *p < 0.5, **p < 0.01, ***p < 0.001.For fluorescence quantification, images were captured using a laser scanning confocal microscope . All the control and experimental fillets were processed in the same way, and the fluorescence images were captured under the same settings for every experimental set. For bouton quantification, CSP-labeled structures were counted at muscle 6/7 of A2 hemisegment. The number of boutons from each NMJ was normalized to the respective muscle area. To calculate the bouton number, NMJs from A2 hemisegment were captured using a plan apochromat 40× objective, 1.4 NA and all the CSP positive boutons were counted manually in ImageJ/Fiji software. For muscle area quantification, images from A2 hemisegment were captured using 20× objective, and the area was quantified using ZEN2 software . For bouton number quantification, the total number of boutons per NMJ was divided by the respective muscle areas. For fluorescence intensity quantification, NMJs from muscle four were captured using a plan apochromat 60×, 1.4 NA objective. For each NMJ, the fluorescence intensity from each bouton was subtracted from the background intensity, and the average intensity was normalized to the control. The fluorescence intensity was calculated using ImageJ/Fiji software. For bouton area quantification, NMJs from muscle 6/7 at A2 hemisegment were captured, and the area was calculated by drawing a free-hand sketch around CSP positive bouton using ImageJ/Fiji software. For multiple comparisons, one-way ANOVA followed by σ2-adaptin mutants caused NMJ overgrowth and upregulation of pMad, an effector of the BMP pathway. Two previously characterized σ2-adaptin alleles, ang7,AP2σ a strong hypomorph and KG02457AP2σ, a null allele was used in the current study , 13, AP2σKG02457/AP2σang7Med , and k00237Mad/+; KG02457/AP2σang7AP2σ  when compared with KG02457AP2σ/ang7AP2σ . However, there was no significant difference between 1118w control, heterozygous KG02457AP2σ/+, and 7tkv/+ , KG02457/AP2σang7AP2σ (B), A12wit, KG02457/AP2σang7AP2σ (C), double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Reducing the levels of BMP Type II receptor in the KG02457/AP2σang7AP2σ background reduces the synaptic overgrowth. Scale bar in C represents 10 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.33 ± 0.03), KG02457/AP2σang7AP2σ (2.50 ± 0.11), and A12wit, KG02457/AP2σang7AP2σ (2.0 ± 0.07). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01, ***p < 0.001. Download Figure 1-1, TIF file.Reducing levels of Wit receptor rescues the bouton number in wit significantly reduced the σ2-adaptin induced synaptic overgrowth , 13Med , and k00237Mad/+; KG02457/AP2σang7AP2σ  in σ2-adaptin mutant background partially restored the bouton area when compared with σ2-adaptin mutant alone ] and Dad [j1E4/+Dad (1.39 ± 0.09)] was comparable with 1118w control (1.26 ± 0.06), transheterozygous j1E4/AP2σKG02457Dad  showed significantly higher bouton number when compared with 1118wcontrols (σ2-adaptin and Dad mutants j1E4/AP2σKG02457Dad (8.51 ± 0.61) when compared with 1118wcontrol  significantly rescued the synaptic overgrowth in σ2-adaptin mutant ang7/AP2σKG02457AP2σ, quantal content (QC), and high-frequency intracellular recording on 1118w control, heterozygous 7/+tkv, KG02457/AP2σang7AP2σ, and 7/+; AP2σKG02457/AP2σang7tkv larvae  were not significantly different from σ2-adaptin mutant KG02457/AP2σang7AP2σ stimulations  and increased size of the SVs in the σ2-adaptin mutants . While there was no change in the number of SVs docked at the active zones , we found significantly decreased thickness of the subsynaptic reticulum  in the σ2-adaptin mutants. These ultrastructural defects were rescued on ubiquitous expression of a σ2-adaptin transgene  or clathrin light chain  in motor neurons showed significantly increased synaptic Tkv levels  or the late endosomal marker, Rab7 (1118w: 100.0 ± 5.71 vs KG02457/AP2σang7AP2σ: 115.6 ± 8.17) were not altered in σ2-adaptin mutant synapses  in σ2-adaptin mutant synapses , KG02457/AP2σang7AP2σ (102.8 ± 11.07), D42-Gal4, ang7AP2σ/KG02457UAS- AP2σ, AP2σ (84.45 ± 6.11), and ex2/93BiRab11 (65.06 ± 7.16). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. *p < 0.05; ns, not significant. Download Figure 6-1, TIF file.Drosophila NMJ  compared with control animals . However, the Tkv enrichment pattern was not the same as that in σ2-adaptin mutants  and presynaptic membrane marker HRP (magenta) in D42-Gal4/UAS-tkv-EGFP  and D42-Gal4/UAS-tkv-EGFP . The intensity profile across the bouton in D42-Gal4/UAS-tkv-EGFP (F) suggests that Tkv is not enriched at the presynaptic membrane. Download Figure 7-1, TIF file.Tkv-EGFP is not localized at the presynaptic membrane in the σ2-adaptin mutants showed reduced Rab11 levels at the NMJ, we next asked whether Rab11 was responsible for the morphologic defects in these mutants. To assess this, we first examined whether reducing Rab11 levels phenocopied σ2-adaptin mutations. Interestingly, we found that Rab11 mutants or neuronal expression of a dominant-negative form of Rab11 (S25NRab11) indeed phenocopied the NMJ morphologic defects in σ2-adaptin mutants and showed NMJ overgrowth  did not prevent the synaptic overgrowth  failed to prevent the synaptic overgrowth in σ2-adaptin mutants , D42-Gal4-driven UAS-Rab11-GFP (B), and D42-Gal4-driven Q70L-GFPUAS-Rab11 (C) double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Expressing the wild-type and active form of Rab11 does not show morphological changes at the synapse. Scale bar in C represents 20 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.56 ± 0.06), UAS-Rab11-GFP/+; D42-Gal4/+ (1.56 ± 0.07), and Q70L-GFPUAS-Rab11; D42-Gal4/+ (1.58 ± 0.09). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. Download Figure 8-1, TIF file.Expressing a wild-type or constitutively active form of Rab11 does not alter the synaptic morphology. 10.1523/ENEURO.0044-22.2022.f8-2Extended Data Figure 8-2σ2-adaptin mutants. A–D, Confocal images of NMJ synapses at muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in (A) Control animals, (B) CAD42-Gal4 > Rab11, (C) KG02457/AP2σang7AP2σ, and (D) CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 double immunolabeled with a presynaptic synaptic vesicle marker, CSP (magenta) and a neuronal membrane marker, HRP (green) to mark the bouton outline. Scale bar in D represents 20 μm. E, Histogram showing average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.05 ± 0.11), CAD42-Gal4 > Rab11 (1.4 ± 0.10), KG02457/AP2σang7AP2σ (2.9 ± 0.14), and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (2.5 ± 0.14). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01; ns, not significant. Download Figure 8-2, TIF file.Constitutively active Rab11 does not restore the NMJ morphological defects of 10.1523/ENEURO.0044-22.2022.f8-3Extended Data Figure 8-3σ2-adaptin. A, Representative traces of mEJP in control, DND42 > Rab11, WTD42 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, DND42 > Rab11, KG02457/AP2σang7AP2σ, and WTD42 > Rab11, KG02457/AP2σang7AP2σ larvae. B, Representative tracesof EJP in control, DND42 > Rab11, WTD42 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, DND42 > Rab11, KG02457/AP2σang7AP2σ, and WTD42 > Rab11, KG02457/AP2σang7AP2σ larvae. C, Quantification of average EJP amplitude in control (44.42 ± 1.93), DND42 > Rab11 (53.89 ± 2.35), WTD42 > Rab11 (52.71 ± 2.42), heteroallelic AP2σKG02457/AP2σang7 (43.57 ± 4.52), DND42 > Rab11, KG02457/AP2σang7AP2σ (38.03 ± 3.60), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (32.92 ± 2.14). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average mEJP amplitude in control (0.56 ± 0.01), DND42 > Rab11 (0.96 ± 0.08), WTD42 > Rab11 (0.78 ± 0.05), heteroallelic KG02457/AP2σang7AP2σ (1.19 ± 0.05), DND42 > Rab11, KG02457/AP2σang7AP2σ (0.13 ± 0.14), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (1.46 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing average mEJP frequency in control (1.38 ± 0.10), DND42 > Rab11 (4.17 ± 0.47), WTD42 > Rab11 (3.78 ± 0.31), heteroallelic KG02457/AP2σang7AP2σ (5.01 ± 0.23), DND42 > Rab11, KG02457/AP2σang7AP2σ (4.33 ± 0.23), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (4.06 ± 0.34). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. F, Quantification of quantal content in control (50.56 ± 5.82), DND42 > Rab11 (53.14 ± 4.91), WTD42 > Rab11 (66.11 ± 5.48), heteroallelic KG02457/AP2σang7AP2σ (38.95 ± 3.65), DND42 > Rab11, KG02457/AP2σang7AP2σ (30.14 ± 3.03), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (25.07 ± 0.93). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. Download Figure 8-3, TIF file.Overexpression of Rab11 does not restore the functional defects of 10.1523/ENEURO.0044-22.2022.f8-4Extended Data Figure 8-4σ2-adaptin mutants. A, Representative traces of mEJP in control, CAD42-Gal4 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 larvae. B, Representative traces of EJP in control (1118w), CAD42-Gal4 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 larvae. C, Quantification of average mEJP amplitude (mV) in control (0.67 ± 0.04), CAD42-Gal4 > Rab11(1.13 ± 0.17), heteroallelic KG02457/AP2σang7AP2σ (1.05 ± 0.09) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (1.30 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average EJP amplitude in control (44.52 ± 1.07), CAD42-Gal4 > Rab11 (56.85 ± 4.221), heteroallelic KG02457/AP2σang7AP2σ (35.40 ± 1.925) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (41.41 ± 1.340). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing quantal content in the control (63.43 ± 4.81), CAD42-Gal4 > Rab11(26.88 ± 4.42), heteroallelic KG02457/AP2σang7AP2σ (47.08 ± 4.13) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (37.38 ± 5.83). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple comparison test. **p < 0.01; ns, not significant. Download Figure 8-4, TIF file.Constitutively active Rab11 does not restore the physiological defects of σ2-adaptin and Rab11 increase Tkv accumulation and BMP signaling to generate similar NMJ phenotypes via independent pathways.In conclusion, Endo, Synj, nwk, shi, Clc, brat, and σ2-adaptin mutant NMJs  are reduced by half in σ2-adaptin mutant synapses.BMP signaling has been extensively studied in the context of neuronal growth in which the activated Tkv is endocytosed and fuse with the early endosomes, where it activates downstream signaling molecules. The signaling is attenuated when these activated receptor-containing vesicles recycle back to the plasma membrane or fuse with lysosomes for degradation . Trafficσ2-adaptin mutant synapses. The intensity profile of Tkv and HRP across the bouton shows that σ2-adaptin mutant has a higher intensity of Tkv at the membrane, indicating that a significant proportion of the Tkv receptors are accumulated at the presynaptic membrane. The pattern of Tkv enrichment in σ2-adaptin and Rab11 mutants were distinct. While σ2-adaptin mutant synapses showed Tkv enriched at synaptic membranes, Rab11 mutants had a rather punctate distribution within the bouton. The differential distribution of these proteins could be because of their distinct roles in the neurons. While σ2-adaptin plays a critical role in retrieving the SV-membrane from the presynaptic membrane, Rab11 is a component of the endolysosomal machinery. A clear separation of Tkv signals/pixels distinguishing plasma membrane Tkv from endosomal Tkv is challenging at the NMJ, given the resolution limit of the confocal system. The difference in Tkv distribution in σ2-adaptin and Rab11 mutants also argues for additional pathways, other than Rab11, through which σ2-adaptin may regulate BMP signaling.Interestingly, levels of early and late endosomes marked with Rab5 and Rab7, respectively, remain unaffected at the σ2-adaptin mutants could be accumulated either at the plasma membrane because of inefficient CME or at the early endosomes caused by inefficient recycling. If Tkv were accumulated at the early endosomes, we would expect greater colocalization with Rab5, which is not the case. However, based on reduced Rab11 staining at the σ2-adaptin mutant synapses, we conclude that the portion of the receptors in Rab5 positive early endosomes fail to recycle back to the plasma membrane. This conclusion also fits with previous observations that defective CME results in the accumulation of endosome-like structures in cultured hippocampal neurons (Drosophila NMJ, endocytosis attenuates BMP signaling (Drosophila wing discs and intestinal stem cells, endocytosis facilitates the signaling cascade by internalizing Tkv (σ2-adaptin mutants fail to recycle remaining receptors from early endosomes leading to enhanced signaling and NMJ growth defects.Tkv receptors in  neurons  and is s neurons . At the ignaling , whereaszing Tkv , pointinwit mutants, the size of the NMJ is significantly reduced with concomitant reduced evoked excitatory potentials (tkv and sax, co-Smad Medea, and transcription factor Mad showed that these NMJs have smaller synapses with severe functional deficits (gbb mutant larvae also exhibit shorter NMJs with severely reduced evoked potentials (σ2-adaptin mutant synapses show a modest reduction in evoked potentials, and the protein is dispensable for maintaining basal synaptic transmission (Endo, Synj, and Dap160, show a rapid stimulus-dependent decline in EJP amplitude that recovers following a period of rest after the high-frequency stimulation paradigm (σ2-adaptin mutants do not recover from synaptic depression even after the 90s rest after the cessation of high-frequency stimulation (1118wcontrols. EJP and high-frequency recordings from σ2-adaptin mutant synapses with one copy of 7tkv did not show any rescue in synaptic function. These data drive the conclusion that partial reduction of BMP pathway components can only rescue morphologic defects in σ2-adaptin mutants but not functional aspects and that morphologic and functional deficits can be discriminated in these mutants. Besides, the partial rescue of bouton size and bouton clustering at the NMJ argues for possible deregulation of multiple signaling pathways in σ2-adaptin mutants that remain to be explored.Morphologic features of synapses often dictate functional outcomes, and physiological analyses of BMP signaling mutants reveal the same. In tentials . Analysedeficits . Consisttentials . σ2-adapsmission . Howeverparadigm . In our mulation . This obDrosophila NMJ. First, we show using genetics that the morphologic defects in σ2-adaptin mutant synapses can be partially rescued by introducing a mutant copy of the BMP receptors, tkv, and wit. We also show a direct epistatic interaction between σ2-adaptin and the inhibitory Smad, Dad. Second, using immunohistochemistry, we show that σ2-adaptin mutant synapses accumulate Tkv at the plasma membrane and some of these receptors that are endocytosed and make it to the early endosomes fail to recycle back to the plasma membrane. Third, our electrophysiology data establish that morphologic and functional defects can be discriminated in σ2-adaptin mutants. Finally, our electron micrographs provide evidence for the presence of large endosomes and support our conclusion that σ2-adaptin is critically required at a later step of vesicle regeneration following endocytosis from the plasma membrane.Our study uncovers and extends the existing knowledge of synaptic growth signaling and endocytosis. We provide four lines of evidence on the critical role of σ2-adaptin in modulating BMP-dependent synaptic growth signaling at the σ2-adaptin mutant synapses that could be restored by neuronal expression of σ2-adaptin transgenes. However, the structural and functional defects of σ2-adaptin mutants could not be restored by neuronal expression of Rab11WT or Rab11CA. This rules out the notion that σ2-adaptin phenotypes result from the observed reduction of Rab11. The Rab11 mutant used in this study is a hypomorph with substantial Rab11 protein being detected by us and others (Rab11 hypomorphic mutants may explain why pMad levels were not as upregulated when compared with σ2-adaptin mutants. Finally, the separation of Tkv immunoreactivity from the synaptic membrane versus that of the endosomal membrane is challenging, given the resolution of the imaging system used here. Our electron micrographs, however, convincingly show an accumulation of endosome-like structures close to the plasma membrane in σ2-adaptin mutant boutons. The endosome-like structures observed in the σ2-adaptin mutants are strikingly similar to structures previously reported for clathrin (While this study does not report a direct biochemical or epistatic interaction between σ2-adaptin and Rab11, we observed a significant reduction in Rab11 immunoreactivity in d others . This prclathrin , AP180 (clathrin , and Rabclathrin , among oDrosophila NMJ. In the absence of AP2, recycling of Tkv is compromised, which results in its enrichment at the presynaptic membrane and/or in the early endosomes leading to elevated BMP signaling and synaptic overgrowth (We propose a model in which σ2-adaptin/AP2-complex is required for the attenuation of BMP signaling at the ergrowth . This st


In order to fabricate homogeneous large-scale Ti-5Al-5V-5Mo-3Cr (Ti-5553) alloy bulk with fine and equiaxial β grain, we performed a series of multi-axial α + β field forging with 62 forging cycles on the large-scale Ti-5553 billet by using 12.5 MN high-speed hydraulic press. The β-annealed microstructure was the starting microstructure of the billet. After the 6th forging cycle, β grain deformed dramatically, and the grain-boundary network developed within the irregular β grain. As the forging cycle increased to 44, the volume fraction of the fine and equiaxial β grain that is less than 20 μm, which is caused by dynamic recrystallization, increased gradually. However, the incomplete dynamic recrystallization region within the original β grain could not be eliminated. As the forging cycle further increased, the volume fraction of the fine and equiaxial β grain did not increase. In contrast, the abnormal grain growth of the β phase occurred during 50th~62nd forging cycle. Here, we attribute the formation of the incomplete dynamic recrystallization region and the abnormal grain growth of the β phase to the high deformation rate of the α + β forging. The refining behavior of β grain and the abnormal coursing β grain, which is found during the multi-cycle multi-axial forging of large-scale Ti-5553 alloy billet, are seldom reported in the isothermal compression of small-scale Ti-5553 alloy specimen. The findings of the paper are instructive for improving the sub-transus forging strategy that is used to fabricate the large-scale homogeneity Ti-5553 alloy billet with fine and equiaxial β grain. Near-β titanium alloys are widely used in the aviation industry because of their high strength-to-weight ratio and excellent corrosion resistance . Based oTi-5Al-5V-5Mo-3Cr (Ti-5553) alloy, which is a relatively new near-β titanium alloy, exhibits several unique merits such as improved hardenability and better compositional homogeneity in melting as compared to other titanium alloys ,7. OwingThe Ti-5553 alloy ingot with the dimension of Φ 260 mm × 840 mm was melted by a consumable electrode vacuum furnace with a maximum melting weight of 1000 Kg. Substances used for melting the ingot of Ti-5553 alloy were supplied by Northwestern Institute for Non-ferrous Metal Research. The aim of the β-field forging is to eliminate the as-cast microstructure of Ti-5553 alloy ingot. The β-field forging was conducted on 12.5 MN high-speed hydraulic press. Before starting each forging cycle, the billet (ingot) was kept in the pre-heated furnace for 180 min. The temperature of the first cycle β-field forging, the second cycle β-field forging and the third cycle β-field forging were 1180 °C, 1080 °C and 900 °C, respectively. In the present work, length, width, and height of the Ti-5553 billet for α + β field are 200 mm, 200 mm, and 400 mm. Before starting the α + β forging, the billet was annealed at 900 °C for 120 min, and was quenched into the water tank, and was then annealed at 785 °C for 120 min. It is hard to retain the microstructure of the billet before the α + β forging to room temperature, because the length, width, and height of the billet are larger than the maximum quenching diameter of Ti-5553 alloy [The slice with the dimension of 200 mm × 200 mm × 12 mm was machined from the billet forged in the β-field, and the cylinder with the dimension of Φ 10 mm × 10 mm was cut from the Ti-5553 alloy slice. To explore the microstructure of the billet before α + β field forging, an annealing treatment was conducted on the cylinder. The cylinder was first annealed at 900 °C for 120 min following water-quenching, and was then annealed at 785 °C for 120 min following water-quenching. The microstructure of the cylinder was examined by Zeiss Axio Vert A1 optical microscope. The observed surface of the metallographic specimen was the top surface of the cylinder. The normal of the observed surface was parallel to the Z direction of the billet.The aim of the α + β field forging is to refine the β grain of the large-scale billet to the grain-size range from 5 μm to 15 μm. α + β field forging of the billet was conducted on a 12.5 MN high-speed hydraulic press. The α + β forging temperature was 785 °C. Before starting each forging cycle, the billet was kept in the pre-heated furnace for 120 min. The multi-axial forging strategy for each cycle was shown in After the 6th forging cycle, the 14th forging cycle, the 22nd forging cycle, the 38th forging cycle, the 44th forging cycle, the 50th forging cycle, the 56th forging cycle, and the 62nd forging cycle, a slice with a thickness of 20 mm was cut from the end of the square billet. The cylinder with the dimension of Φ 10 mm × 10 mm was cut in the center of each slice, in order to analyze the β grain size of the billet.In our previous work, we found that after the 5 min aging at 700 °C the α precipitation forms on the β grain boundary and does not form inside the β grain . MicrostIn order to investigate the role of the forging cycle in the β-grain size of Ti-5553 alloy billet and to characterize the grain boundary of β phase, the solution-ageing treatment was performed on the Φ 10 mm × 10 mm cylinder. The solution was carried out at 785 °C for 1 h following water-quenching, and the short-time ageing was carried out at 700 °C for 5 min following water-quenching. The microstructure of the Ti-5553 alloy cylinder was examined by Zeiss Axio Vert A1 optical microscope. The observed surface of the metallographic specimen was the top surface of the cylinder. The normal of the observed surface was parallel to the Z direction of the billet.By using the Gleeble-3800 testing machine, isothermal compression test was conducted on the alloy after the 44th cycle forging and the 62nd cycle forging. The cylinder with the dimension of Φ 8 mm × 12 mm was machined from the slices that were cut from the billet after the 44th cycle forging and the 62nd cycle forging, respectively. The K-type thermocouple was welded on the mid-height side of the specimen to monitor the temperature. To decrease the friction between the surface of the compression plate and the top surfaces of the Ti-5553 alloy cylinder, the graphite grease and the tantalum film were used. Under the clamping load of 1000 N, the specimen was heated to the target temperature at the heating rate of 6 °C/s. The specimens were compressed at 785 °C. Once at the target temperature, the specimen was held for 180 s to achieve thermal equilibrium. The strain rate of the isothermal compression test was 0.1/s. The isothermal compression strain was 0.69. After finishing the deformation, the specimen was air-cooled to room temperature. All the compressed samples were annealed at 785 °C for 1 h following water-quenching, and were then annealed at 700 °C for 5 min following water-quenching. The metallographic preparation of the heat-treated sample was carried out by sectioning it in half, embedding it in the resin, and grinding-polishing-etching techniques. The microstructure in the center of the specimen was investigated by Zeiss Axio Vert A1 optical microscope.2 μm. GB) has precipitated on the grain boundary of prior β grain, and intragranular α (αIntra) has precipitated inside the prior β grain. Some prior β grains process the high volume fraction of αIntra, while the volume fraction of αIntra is considerably low in the other prior β grains. In addition, the precipitation-free zone could be found in the grain boundary region. In summary, we conclude that the microstructure of the cylinder should be the typical β-annealed microstructure.Intra. As a result, the microstructure of the large-scale Ti-5553 alloy billet should also be the lamellar microstructure before starting the multi-cycle and multi-axial forging in α + β field.In the present work, the ex situ heat-treatment was conducted on the Φ 10 mm × 10 mm Ti-5553 alloy cylinder that was machined from the large-scale Ti-5553 alloy billet forged in β field, and the heat-treatment parameters of the cylinder were the same as those of the large-scale Ti-5553 alloy billet. Although the heating rate of the cylinder is different than that of the large-scale billet, it hardly affects the size of the prior β grain and the volume fraction of α2μm before the α + β forging ( forging a. HoweveAlthough 44 cycles of multi-axial forging has been conducted, the incomplete recrystallization region cannot be eliminated. As a result, the isothermal compression test was performed to verify the forging strategy. In order to eliminate the coarse β grain, which is caused by the abnormal grain growth, of the billet after the 62nd multi-axial α + β forging, isothermal compression was conducted. The aim of the present work is to fabricate the homogeneous large-scale Ti-5553 alloy bulk with equiaxial β grain that is less than 20 μm by multi-cycle and multi-axial forging in α + β field. However, the results contradict the original hypothesis. First, although the coarse β grain, which is retained after the β-field forging, could be refined gradually from the 6th forging cycle to the 44th forging cycle, the incomplete recrystallization region that processes un-recrystallized β grain could not be eliminated utterly. Second, the abnormal grain growth of β grain occurs during the 50th~62nd forging cycle. These data suggest our forging strategy, including forging temperature, forging strain, or forging strain rate, might be inappropriate.As pointed out by Weiss, the recommended α + β forging temperature for near-β titanium alloys is in the temperature range that is 30–50 °C below the β-transus temperature . In the In our previous work, we investigated the effect of the compression strain on the dynamic recrystallization behavior for the β phase of Ti-5553 alloy during the sub-transus thermomechanical processing. Our findings suggest that in the proper forging temperature range the critical strain for the dynamic recrystallization β phase is 0.30 . The mecIn the present work, the strain distribution of the large-scale Ti-5553 alloy bulk during multi-cycle and multi-axial forging is more complex than that of isothermal compression. However, the dynamic recrystallization mechanism of the β phase during the engineering forging should be the same as that of the isothermal compression, which is dominated by the formation of the new grain boundary. As mentioned above, the incomplete recrystallization region of the β phase is still present in the bulk even after the 44th cycle forging cycle. It is understandable that the accumulated forging ratio of the bulk is considerably high after the 44th cycle forging cycle, and the accumulated strain for each part of the bulk should be much higher than the critical strain for the dynamic recrystallization β phase. Therefore, we conclude that the retained incomplete recrystallization region of the large-scale Ti-5553 alloy bulk is not caused by the insufficient forging strain.In the present work, the multi-cycle and multi-axial α + β field were performed at 785 °C. Before starting each forging cycle, the billet was annealed at 785 °C for 120 min. As mentioned in First, the high forging strain rate tends to result in a drastic temperate rise in each α + β forging cycle. It is reported that the temperature rise of β titanium alloys might be higher than 45 °C during the α + β isothermal compression as the compression strain rate reaches 10/s . As a reSecond, the drastic temperate rise during α + β forging might lead to the dynamic α→β transformation of the billet. Li et al. reported the dynamic α→β transformation of Ti-5Al-5V-5Mo-1Cr-1Fe β titanium alloy, the chemical composition of which is considerably similar to that of the Ti-5553 alloy, could occur during the hot compression in α + β field . InteresIn summary, the temperature rise and the dynamic α→β transformation, which is caused by the high strain rate forging, contribute to the formation of the incomplete recrystallization β region and the abnormal β-grain growth. Furthermore, we will investigate the role of forging strain rate in β grain size inhomogeneity of large-scale Ti-5Al-5V-5Mo-3Cr alloy billet by using the hydrostatic press.In this paper, we conducted a series of multi-axial forging with 62 forging cycles on large-scale Ti-5553 billet and investigated the β grain size evolution of billet during the whole α + β forging series. The following conclusion could be drawn.It is impossible to fabricate large-scale homogeneous Ti-5553 alloy billet with fine and equiaxial β grain by multi-cycle α + β forging with a high deformation rate.The inappropriate forging parameters, especially the deformation rate, might lead to the incomplete dynamic recrystallization of the β phase and abnormal grain growth of β phase during the multi-cycle and multi-axial forging of the large-scale Ti-5553 alloy billet.After performing the solution treatment at 785 °C and the 5 min ageing treatment at 785 °C, the β grain size and β grain shape of Ti-5553 alloy forging billet could be characterized by an optical microscope.


The beeswax used in Chinese traditional hot waxed wood technology has several drawbacks, such as high price, scarce resources, and poor heat resistance. To expand the application of hot waxing technology in the field of wood decoration and protection, polyethylene wax was modified by a grafted maleic anhydride-methyl methacrylate co-monomer. A type of modified polyethylene wax with low cost, high melting point, high stability, and strong polarity suitable for hot waxed wood was prepared as a replacement for beeswax. The effects of the grafting conditions on the chemical properties, thermal properties, chemical structure, and crystallization properties of modified polyethylene wax were studied and compared to those of beeswax. The results show that maleic anhydride-methyl methacrylate co-monomer grafting can effectively improve the acid value of polyethylene wax. For the ratio of two monomers of 1/1, the total amount of monomer of 8 wt%, the amount of initiator of 2 wt%, the reaction temperature of 150 °C, and the acid value of modified polyethylene wax was consistent with that of beeswax, realizing the simulation of the main chemical properties of beeswax. The thermal stability and melting temperature of the modified polyethylene wax are significantly higher than those of beeswax, its crystal structure is similar to that of beeswax, and the cyclic anhydride groups and ester groups introduced by co-monomer grafting endow it with polar groups that play an important role in the wood hot waxing process. Wood decoration by hot waxing with beeswax has long been used in China and is a simple and environmentally friendly method for the surface waterproofing treatment of hardwood furniture. Although the beeswax used in the traditional hot waxed wood process has the advantages of good gloss and good compatibility, its wide use in modern industrial production is hindered by its high cost and shortage of resources . In addi2) groups, the compatibility of polyolefin with other polar polymers is enhanced. MAH has been successfully grafted onto polyolefin molecular chain by solution [Grafting is considered to be an important technology for modifying the physical and chemical properties of polymers. Free radical or anionic grafting methods are used in the grafting modification of polymers ,9. Free-solution ,21,22,23solution ,27,28,29solution ,31. In aHowever, the grafting degree of MAH on polyolefins is usually quite low. This is due to the low reactivity of MAH to free radicals and its low solubility in the polyolefin matrix. The low free-radical reactivity of MAH is due to its intrinsic structural symmetry and electron density deficiency around the double bond . To obta1H-NMR), and X-ray diffraction (XRD). The effects of the ratio of grafting monomers, the number of monomers, the amount of initiator, and reaction temperature on the properties of the product and the grafting mechanism were investigated. Based on these investigation and comparison with the related properties of beeswax, the modified polyethylene wax with high melting point, high stability, and strong polarity suitable for hot waxed wood process was prepared as a replacement for beeswax.In this study, methyl methacrylate (MMA)-assisted grafting MAH was selected to modify polyethylene wax. Because of the low reactivity ratio of MMA and MAH , it easiBeeswax (BW) was taken from the Dengfeng Bee Farm in Henan Province, China. Samples were taken from the center of the wax block and heated and melted in distilled water to filter impurities. Polyethylene wax (PEW), produced by Thailand SCG, Co. , was dissolved and refluxed with xylene at 120 °C for 2 h, and then methanol was added to precipitate and purify it. Analytical grade maleic anhydride (MAH) was purchased from Tianjin Guangfu Fine Chemical Research Institute . Analytical grade methyl methacrylate (MMA) was purchased from Aladdin Reagent Co., Ltd. . Analytical grade benzoyl peroxide (BPO) with a half-life time of 1 min at 131 °C was purchased from Shanghai McLean Biochemical Technology Co., Ltd. . Analytical grade xylene and methanol were provided by Tianjin Fuyu Fine Chemical Co., Ltd. . The above reagents were used without further purification. −1 was carried out to obtain nitrogen atmosphere for the four-port bottle. A certain mass of purified polyethylene wax and xylene with twice the mass of polyethylene wax were poured into a four-port bottle and vibrated several times for full mixing of polyethylene wax and xylene. The four-port bottle was poured into a constant temperature oil bath and connected to a reflux condensing tube, a thermometer, a slurry electric agitator, and gas conduit. Then, a nitrogen purge with a nitrogen flow rate of 20 mL minThe oil bath was heated to 120 °C and stirred at a constant speed of 170 rpm to ensure that the polyethylene wax was completely melted and mixed with xylene. Then, the pre-prepared BPO initiator and MAH/MMA xylene solution were added dropwise to the polyethylene wax/xylene mixture stirred at constant speed (360 rpm) using a constant-pressure separation funnel, and the mixture reacted for 3 h at 130–160 °C. The details of the procedure and grafting temperatures are shown in g-MAH-MMA) was obtained by vacuum drying at 50 °C for 12 h. The reaction equation for the grafting of MAH-MMA onto PEW is shown in At the end of the reaction, the reaction solution was agitated rapidly, and the crude product was precipitated with methanol. The mass of methanol was three times higher than the mass of polyethylene wax. After methanol washing and filtration, the crude product was soaked in xylene for 24 h to extract the unreacted MAH/MMA monomers, MAH/MMA homopolymers, and other impurities. Purified graft maleic anhydride-methyl methacrylate modified polyethylene wax  was placed into a 250 mL flask, xylene solution (40 mL) was added, and the sample was refluxed and dissolved in an oil bath until the sample was clear and transparent. Three to five drops of phenolphthalein solution were added and the solution was titrated to pink with a standard KOH solution (0.05 mol/L) with the color not fading within 10 s indicating the titration end point. The flask was shaken violently during titration and the sample was reheated if precipitation occurred during the titration. The number of milliliters of KOH standard solution used for titration was recorded and a blank test was performed at the same time. The acid value (KOH mg/g) was calculated by Equation (1). The experiments were repeated five times for each group of wax samples, and the average value was taken as the final result.A is the milliliters of the KOH standard solution required for titration of the sample, B is the milliliters of KOH standard solution required for titration of the blank sample, N is the normality of KOH standard solution, and C is the grams of the tested wax sample. The acid values of BW, PEW, and PEW-g-MAH-MMA were determined using a Universal Test Machine . The samples were cut into strips with dimensions of 1 × 8 cm and conditioned at 23 °C and 50%RH for at least 24 h. The samples were tested at a crosshead speed of 10 mm·min−1. Each sample was repeated 5 times, and the average value was taken as the final result.According to the ASTM D882-02 standard, the tensile strength and elongation at break of PEW and PEW-g-MAH-MMA was carried out using a thermogravimetric analyzer  to characterize the thermal stability of different wax samples. The sample (5–10 mg) was weighed each time and heated from 30 °C to 600 °C at a heating rate of 10 °C min−1 under nitrogen atmosphere (50 mL min−1). The derivative mass loss (DTG) curves were obtained, and the thermal stability of the wax sample was evaluated by the temperature corresponding to the maximum thermal weight loss from the DTG curve. The thermogravimetric analysis of BW, PEW, and PEW-g-MAH-MMA samples were studied using a DSC-60 differential scanning calorimeter . The sample weight was 5–10 mg, and the experiments were performed in nitrogen atmosphere. The test procedure is divided into three steps: (1) heating from 25 °C at a heating rate of 10 °C min−1 to 160 °C and equilibrating for 5 min to eliminate the thermal history; (2) naturally cooling the temperature from 160 °C to 25 °C; and (3) reheating to 160 °C at 10 °C min−1. The reheating DSC curves were recorded to study the thermal behavior of the samples. The thermal properties of the BW, PEW, and PEW-g-MAH-MMA samples were analyzed by Fourier transform infrared spectroscopy. The spectra were collected with an average of 32 scans at a resolution of 4 cm−1 in the wavenumber range of 400–4000 cm−1 in the transmittance mode. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was performed on a Nicolet is50 FTIR spectrometer . The active functional groups of BW, PEW, and PEW-1H-NMR) spectroscopy was recorded at 600 MHz operating frequency using 1H NMR spectrometer  at 120 °C.PEW and PEW-g-MAH-MMA samples were prepared in tetrachloroethane solution and sealed in NMR tubes. Proton nuclear magnetic resonance  using Cu target Kα radiation, an X-ray wavelength of 0.154 nm, a radiation tube voltage of 40 kV, a tube current of 40 mA, a scanning angle range of 10–50°, a scanning speed of 4°/min, and a step of 0.02°. The degree of crystallinity of sample was calculated by dividing the total area under Bragg peaks on the total area under Bragg peaks plus the area under hump.The crystallization properties of the BW, PEW, and PEW-−1 (shoulder peak at 2955 cm−1) and 2848 cm−1 are due to the asymmetric and symmetric C-H stretching of aliphatic methylene (-CH2-) and methyl (-CH3). The absorption peaks near 1473 cm−1 and 1463 cm−1 are due to the bending vibrations of -CH3 and -CH2-. The absorption peaks near 730 cm−1 and 719 cm−1 are attributed to the in-plane wagging vibrations of -CH3 and -CH2-. The peak at 1732 cm−1 is due to the stretching vibration of the unconjugated carbonyl groups in esters, the peak at 1711 cm−1 arises from the vibrations of unconjugated carbonyl groups in free fatty acids, and the peak at 1170 cm−1 is due to the C-O vibration in esters. Similar FTIR spectra of BW have been reported in the literature [terature ,39,40,41−1). Clear differences between the spectra of PEW and BW are observed in the fingerprint region. There is no absorption band related to esters and free fatty acids in PEW. Different from the FTIR spectrum of PEW, in the FTIR spectrum of PEW-g-MAH (−1 are observed. The peaks at 1861 cm−1 and 1782 cm−1 are attributed to the asymmetric and symmetric stretching vibrations of cyclic anhydride carbonyl (C=O) [−1 is attributed to the stretching vibration of the carbonyl groups in the carboxylic groups [g-MAH, the FTIR spectrum of PEW-g-MAH-MMA  ,43,44, ac groups . These rs on MMA . It was 1H-NMR spectra of PEW-g-MAH-MMA with the corresponding PEW, and further analyzes the structure of MAH-MMA grafted PEW by 1H-NMR. Compared with PEW, PEW-g-MAH-MMA has two new chemical shifts at 3.7 and 3.2 ppm. 3.2 is the proton peak of methylene (-CHCH) in MAH structural unit, and 3.7 belongs to the proton peak of methyl hydrogen on -COOCH3 in the MMA structural unit [1H-NMR spectrum belong to the proton peaks in the xylene solvent and have not been completely removed from the product. 1H-NMR analysis further proved that MAH and MMA formed a copolymer and grafted onto PEW.ral unit ,47, indig-MAH-MMA similar to those of BW, acid value was selected as the key indicator of the performance of PEW-g-MAH-MMA. Due to the hydrolysis of maleic anhydride (MAH) to maleic acid, PEW-g-MAH-MMA also showed acidity. Based on a large number of previous experiments, the acid value of PEW-g-MAH-MMA is affected by the formulation parameters and process conditions. This section studies the relationship between the acid value of PEW-g-MAH-MMA and the weight ratio of MAH/MMA, the total amount of monomer, the amount of initiator (BPO), and reaction temperature. The results are shown in According to the physical and chemical requirements for BW products in the GB/T 24314-2009 national standard, the acid value is the most important chemical property of BW that directly reflects the polarity and advantages and disadvantages of BW products. To make the chemical properties of PEW-g-MAH-MMA is shown in g-MAH-MMA increases gradually. The maximum acid value is obtained for the MAH/MMA weight ratio of 1/1. When the MMA content was higher than that of MAH, the acid value decreased gradually, and when the weight ratio of MAH/MMA was 1/4, the acid value decreased sharply, and was even lower than that of the MAH grafted alone. Because of its symmetrical structure and the low electron density around the -CH=CH- bond [g-MAH-MMA was significantly increased by the addition of MMA. It is observed from the g-MAH-MMA, and the mechanism of MMA-assisted MAH grafting PEW depends on the MAH/MMA ratio.The effect of the weight ratio of MAH and MMA on the acid value of PEW-CH- bond ,48, MAH CH- bond , the intg-MAH-MMA is reached when the MAH/MMA ratio reaches 1/1. (2) For the MAH/MMA ratio > 1/1, a fraction of MAH forms a copolymer with MMA. The MAH/MMA copolymer is seldom generated because of the low MMA content. Therefore, the grafting form of the PEW macromolecular chain is mainly a single MAH and MAH-MMA copolymer graft. Therefore, for the MAH/MMA ratio of 1/0.5, the acid value of PEW-g-MAH-MMA is between those of MAH/MMA (1/0) and MAH/MMA (1/1). (3) For the MAH/MMA ratio less than 1/1, some MMA monomers react with MAH to form MAH-MMA copolymers, while excess MMA monomers will preferentially react with PEW macroradicals, consuming primary radicals and PEW macroradicals and resulting in a decrease in the amount of grafted anhydride and the acid value. In addition, with the further increase in the MMA content, due to the easy self-polymerization of MMA [g-MAH-MMA is lower than that of the MAH grafted alone. (1) Because of the similarity of the molar mass of MAH and MMA, for the addition of MAH and MMA with the same molar amount , the two monomers can easily interact with each other. Theoretically, all MAH and MMA can form MAH-MMA copolymers, thus forming a longer branching structure on the PEW macromolecular chain and resulting in an increase in the number of grafted anhydrides. Therefore, the highest acid value of PEW-n of MMA ,49, the g-MAH-MMA. With the increase in the amount of the monomer, the acid value increases. When the total amount of monomer is in the range of 4–8 wt%, the acid value increases greatly. When the amount of monomer is greater than 8 wt%, a smaller increase in the acid value is obtained. However, with the further increase in the amount of monomer, when the amount of monomer reached 32 wt%, the acid value of PEW-g-MAH-MMA clearly decreased.A MAH/MMA ratio of 1/1 was applied for other formulations because it gave the highest acid value. g-MAH-MMA is low. The increase in the amount of the monomer helps to increase the likelihood of the collision with macroradicals and fully exploits the radical initiation of monomer grafting. Therefore, the acid value of PEW-g-MAH-MMA increases with increasing monomer content. However, a too high monomer content may give rise to the shielding effect and reduce the initiation efficiency of the radicals. In addition, a too high monomers content will increase the probability of side reactions, such as homopolymerization; reduce the amount of actual grafted anhydride; and lead to a decrease in the acid value. The process of radical grafting actually starts with the decomposition of BPO to form primary radicals that then initiate PEW macroradicals by hydrogen abstraction . Then, mg-MAH-MMA is shown in g-MAH-MMA increases at first and then decreases slowly with the increase of the amount of BPO. When the amount of BPO reaches 2 wt%, the acid value of PEW-g-MAH-MMA is the highest. For a small amount of BPO, the number of radicals in the grafting reaction system is insufficient to initiate the grafting reaction, resulting in a small number of grafted anhydride and a low acid value. With the increase in the amount of BPO, the number of primary radicals increased, so that more PEW macroradicals were produced by the hydrogen extraction reaction, which is beneficial for the full initiation of the graft polymerization of MAH-MMA monomers. The effect of the content of BPO on the acid value of PEW-g-MAH-MMA. However, the amount of BPO is not only directly related to the number of radicals but also related to the degree of cross-linking of the PEW molecules [g-MAH-MMA.Therefore, increasing the amount of BPO in a certain range is an effective approach for increasing the acid value of PEW-olecules ,51. Withg-MAH-MMA. The acid value of PEW-g-MAH-MMA increases with increasing reaction temperature, and reaches the maximum at 150 °C. However, after the acid value reaches the maximum, the further increase of reaction temperature does not increase the acid value of PEW-g-MAH-MMA. On the contrary, the acid value decreased clearly at 160 °C. The process of thermal decomposition of BPO for the production of primary radicals is the initial and the most critical stage of the grafting reaction. The reaction temperature is closely related to the decomposition rate  of BPO [) of BPO ,41,51. Ag-MAH-MMA.With increasing temperature, the rate of BPO decomposition increases, its half-life shortens, and the number of the reaction active centers increases, thus leading to the formation of more macroradicals. In addition, an increase in the temperature is beneficial for the movement of radicals and increases the collision probability of the macroradicals with grafted monomers and leads to the increase in the acid value. However, when the temperature is too high, the BPO decomposition rate is too high and its half-life is too short, resulting in a sharp increase in the number of macroradicals in a short time, thus aggravating the cross-linking of PEW molecules. Due to the influence of steric hindrance, the activation energy of the cross-linking reaction is higher than that of the grafting reaction, making the cross-linking reaction more sensitive to temperature. At higher temperatures, the aggravation of the cross-linking reaction will consume some macroradicals, which is unfavorable for the grafting reaction. In addition, a too high temperature may also lead to the increase in the chain transfer constant of the solvent and the decrease of the initiation efficiency of BPO, leading to the decreased acid value of PEW-g-MAH-MMA with different grafting conditions shown in g-MAH-MMA reaches 18 KOH mg/g, which is consistent with BW and conforms to the national standard. The following characterization and testing of PEW-g-MAH-MMA samples, unless otherwise specified, were performed on the samples prepared with these parameters and process conditions. According to the acid value test results of BW and PEW-g-MAH, and PEW-g-MAH-MMA. Compared with PEW, the tensile strength and elongation at break of PEW-g-MAH and PEW-g-MAH-MMA decreases only slightly, and the difference of mechanical properties between samples is not significant, indicating that the introduction of MAH and MAH-MMA has no obvious effect on the mechanical properties of polyethylene wax.g-MAH, and PEW-g-MAH-MMA as a function of temperature. It is observed that the maximum thermal weight loss temperatures of BW and PEW are 400 °C and 450 °C, respectively. The results for the thermal decomposition and volatilization of BW and PEW indicate that the thermal stability of PEW is higher than that of BW. Compared to PEW, the maximum thermal weight loss temperature of PEW-g-MAH shifted to the high temperature and reached the maximum thermal weight loss at 455 °C. In addition, the DTG curve of PEW-g-MAH also showed a broad peak at 316 °C, which may be attributed to the thermal decomposition of the PEW-grafted MAH monomer.g-MAH, the temperature corresponding to the maximum thermal weight loss of PEW-g-MAH-MMA increased and reached the maximum thermal weight loss at 462 °C. In addition, the peak at approximately 348 °C is attributed to the thermal decomposition of the MAH-MMA co-monomer grafted with PEW. It is clear that the effect of the MAH-MMA co-monomer grafting on the thermal stability of PEW is higher than that of the MAH grafting alone, which may be due to the increase in the grafting amount of MAH by the addition of MMA. In addition, the increase in the number of anhydride groups is beneficial for enhancing the interaction between the anhydride groups and may hinder the decomposition and volatilization of the monomers to some extent. It was found that MAH grafting improves the thermal stability of PEW. This may be attributed to the following effects. First, the introduction of the MAH monomer into the macromolecular chain of PEW increases the molecular weight of PEW and generates a certain number of branched chains on the macromolecular chain of PEW that hinder the thermal movement of the PEW molecular chain to some extent. Second, the five-membered ring structure of the grafted MAH monomer increases the rigidity of the PEW molecular chain, interfering with thermal degradation and improving the heat resistance of PEW. Compared to the DTG curve of PEW-m1 and Tm2) of PEW were significantly higher than those of BW and were found to be 104 °C and 115 °C, respectively. The first peak is related to the solid phase transition of the structure of PEW, and the second peak is related to the melting transition [g-MAH and PEW-g-MAH-MMA are similar to that of PEW, but the melting peak temperature is slightly lower than that of PEW, and Tm1 and Tm2 decrease by 2–4 °C. The difference in the melting peak temperature between PEW-g-MAH and PEW-g-MAH-MMA is not significant, indicating that the existence of MMA has no obvious effect on the melting behavior of MAH-grafted PEW. The decrease in the melting peak temperature may be due to the following reasons. First, the grafting of the anhydride groups on the PEW macromolecular chain increases the amorphous region in the PEW. Motaung et al., have obtained similar findings in the study of MAH grafted low density polyethylene [Similar to the DSC curve of BW, double melting peaks were also observed in the DSC curve of PEW b. Howeveansition ,54. The ethylene . Second,g-MAH, and PEW-g-MAH-MMA. From the XRD curve of BW . These results show that the grafted MAH or MAH-MMA is mainly amorphous, and the increase in the number of disorder features may lead to the decrease in the local ordering of PEW that reduces the crystallinity of PEW-g-MAH and PEW-g-MAH-MMA, in agreement with the results of DSC analysis. Compared to PEW, the XRD curves of PEW-g-MAH-MMA under different grafting conditions shows that for the MAH/MMA ratio of 1/1, the addition of MMA can effectively improve the reaction activity of MAH, increase the extent of anhydride grafting, and significantly increase the acid value. In addition, with the increase in the total amount of the monomer, the amount of BPO, and the reaction temperature, the acid value of PEW-g-MAH-MMA first increased and then decreased. The optimal formulation parameters and process conditions of the PEW-g-MAH/MMA system were determined and were the total amount of the monomer of 8 wt% and 2 wt% BPO, and the reaction temperature was 150 °C for the MAH/MMA ratio was 1/1. For these parameters, the acid value of PEW-g-MAH-MMA reached 18 KOH mg/g, which was consistent with beeswax and in accordance with the requirements set by the national standard, simulating the main chemical properties of beeswax. TGA and DSC analyses showed that the thermal stability and melting temperature of PEW-g-MAH-MMA were significantly higher than those of beeswax. The thermal stability of polyethylene wax was improved by MAH/MMA co-monomer grafting, but the melting temperature decreased slightly. FTIR and 1H-NMR analyses confirmed that MAH and MMA were copolymerized and grafted onto the macromolecular chain of polyethylene wax. The introduction of cyclic anhydride groups and ester groups endowed polyethylene wax with polar groups. XRD analysis showed that the crystal structure of PEW-g-MAH-MMA was similar to that of beeswax, and the grafted MAH-MMA was mainly amorphous. The crystallinity of polyethylene wax decreased slightly with the increase in the number of disorder features. This work developed an effective method for the preparation of modified polyethylene wax that simulates the chemical properties, chemical structure, and crystallization properties of beeswax, and can be used instead of beeswax in the wood hot waxing process, reduce the wax cost of hot waxing process, and meet the industrial demand of batch production. Considering the complexity of hot waxing wood used in outdoor environment, giving more functionality to polyethylene wax in future research should be considered.A modified polyethylene wax with high melting point, high stability, and strong polarity suitable for the hot waxed wood process was prepared by MAH/MMA double monomer grafting method. The study on the acid value of PEW-


NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein. FASFAST is distinguished from other FAPs for several reasons, including the small size 14 kDa), high overall brightness, and ability to bind a variety of fluorogens with various light absorption and emission properties  kDa, hig. The iniFinally, analysis of the FAST structure in the apo form revealed that the first 30 amino acids form two α-helices in the FAST/fluorogen complex but are unstructured in the absence of ligand . ConsideIn this work, we posed several tasks. First, we resolved the structure of nanoFAST without the ligand to understand the effect of the N-terminal part of FAST on the structure and stability of its C-terminal domain. Next, we determined the structure of the nanoFAST/HBR-DOM2 complex to find the key features of the ligand, which make it active in complex with the FAP, while other ligands do not work. Finally, we characterized the internal mobility of the protein backbone to assess the stability of the protein.The structure of the nanoFAST apo state was determined using the conventional NMR approach and was based on NOE distances, chemical shifts, and J-couplings; the high overall quality of NMR data allowed obtaining the almost complete chemical shift assignment . The staWhile the structures of the two proteins are almost identical, the drastic difference between the behavior of FAST and nanoFAST becomes evident starting from the first view of the NMR spectra of nanoFAST . The speAs a second step, we determined the structure of nanoFAST in complex with its fluorogen HBR-DOM2 under the same conditions as the apo form. The quality of NMR spectra was almost perfect , which aAn interesting aspect of the obtained structure is the ionization state of HBR-DOM2. This fluorogen has two chemical groups that can possibly be ionized, the hydroxyl group of the phenolic ring and the amide group of rhodanine. As shown previously, the hydroxyl group of HBR-DOM2 is deprotonated inside nanoFAST , which a13Cα, which depend most on the protein secondary structure, are observed for the residues F2, R26, P42, and G43. , while N871b has a pyridine moiety. These two moieties are packed against the R26 (R52 in FAST) sidechain and alter the position of residues 25–27 and an adjacent turn of the 3–10 helix . The secand G43. . To summ15N relaxation parameters of nanoFAST both in the apo state and in complex with the fluorogen (2), describing the amplitudes of ps-ns motions of the protein backbone and Rex and contributions of slow µs-ms motions to the transverse relaxation. A direct comparison of nanoFAST apo and nanoFAST/HBR-DOM2 reveals that the internal mobility patterns are almost identical. The difference is observed only for the residues 20–30: this region is mobile in nanoFAST apo state, as follows from the decreased NOE and S2 values, and agrees with the obtained spatial structure. In contrast, the region is rigid in the HBR-DOM2 complex, with some slow motions detected for the residues 18–26 (H3) and 29, which can be explained by the formation of an H3 helix. A comparison of dynamics of nanoFAST/HBR-DOM2 and FAST/N871b complexes -glucose were used as nitrogen and carbon sources in the M9 medium. The cells were resuspended in a lysis buffer  and disrupted by ultrasonication. The target proteins were purified by IMAC  and SEC (1× PBS buffer) chromatography. For NMR applications, the target proteins were dialyzed against the NMR-buffer  overnight at 4 °C and concentrated up to 1 mM by ultrafiltration .FAST and nanoFAST proteins were synthesized as described earlier . Brieflyg for 5 min, and the supernatant was placed into the 5 mm NMR tube.To study the structure of a nanoFAST/HBR-DOM2 complex, the ligand was added to the protein sample as an aliquot of 50 mM solution in DMSO-d6  at 1.1× molar excess. The resulting solution was centrifuged at 5000× 3JCCo and 3JNCo couplings were measured from the spin-echo difference constant-time HSQC spectra  were determined by spectrofluorometric titration of protein by chromophore solutions with various concentrations on the Tecan Infinite 200 Pro M Nano dual mode plate reader. The protein concentration was 0.10 µM. The least squares fit (line) gave the dissociation constants KD presented as mean ± SD  in a complex with fluorogens and the apo state. We show that the absence of the N-terminal domain does not affect the structure of the remaining part of the protein. The observed differences between the FAST and nanoFAST are explained by the individual properties of the taken fluorogens. Our data suggest that the N-terminus of FAST destabilizes the dynamics of the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of FAST and nanoFAST interactions with the HBR-type ligands and explains the ligand specificity. nanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted HBRs, which allows the non-canonical intermolecular CH–N hydrogen bonding and provides the optimal packing of the ligand within the hydrophobic cavity of the protein.