Cold-stored Hami melon is susceptible to chilling injury, resulting in quality deterioration and reduced sales. Pre-storage treatment with chitosan reduces fruit softening and chilling injury in melon; however, the underlying mechanism remains unclear. In this study, Gold Queen Hami melons were treated with 1.5% chitosan solution for 10 min before cold storage at 3°C and then the effect of chitosan was examined on fruit firmness, weight loss, chilling injury, soluble solid content (SSC), pectin, and soluble sugar contents of melon fruit. Also, the enzyme activities and gene expressions related to fruit softening and starch and sucrose metabolism were investigated. Chitosan treatment reduced the fruit softening and chilling injury, maintained the high levels of starch and sucrose contents, and regulated the enzyme activities and gene expressions related to starch and sucrose metabolism. Fruit firmness was significantly positively correlated with sucrose and starch contents. Altogether, we uncovered the potential mechanism of chitosan coating mitigating melon softening and chilling injury through the regulation of starch and sucrose metabolism. Hami melon, with its unique flavor, sweet taste, and high nutritional value, is a popular horticultural crop in Xinjiang, Northwest China. The melon fruit is normally harvested in a high temperature and high humidity season, which promotes faster fruit ripening and softening, and in turn reduces the storage life of melon fruit . SofteniRecent studies showed that edible coatings can preserve fruit quality and prolong the postharvest shelf life of cold-stored fruit by regulating the internal gas environment, reducing water loss, and delaying fruit ripening and senescence . ChitosaStarch and sucrose metabolism not only affect fruit quality and sweetness but also are closely related to chilling tolerance during cold storage . SucroseStarch has multiple cellular functions, including in abiotic stress response . In planAt present, much remains to be determined concerning the regulation mechanism of chitosan treatment in melon fruit, including whether the effect of chitosan to mitigate chilling injury is related to starch and sucrose metabolism. Accordingly, we investigated the effects of chitosan treatment on fruit quality indices and starch and sucrose metabolism with the objective of providing a new perspective on the mechanism of chilling injury in melon fruit, and developing an eco-friendly, safe, and efficient postharvest technology to preserve the quality and prolong the shelf life of harvested melons.Cucumis melo L.) melons were picked from a farm in Shihezi, Xinjiang, China at commercial maturity (about 13% SSC). Fresh fruits of uniform shape, size, and color were selected and transported to the laboratory immediately after harvest.“Gold Queen”  was obtained from Rongna Biological Technology Co., Ltd. . Chitosan solution  was prepared as described in The selected melons were randomly divided into two groups; the treatment group was immersed in 1.5% chitosan solution for 10 min, while the control group was treated with distilled water. The treated melons were air-dried and then stored at 3°C and 85-95% relative humidity (RH) for 30 d. Fruits were sampled following the method of th, 12th, 18th, 24th, and 30th d). Weight loss (%) was calculated as follows:Fruit firmness  and chilling injury (%) were measured according to the method of SSC (%) was measured using a digital saccharimeter .Cell-wall components including WSP, ionic-soluble pectin (ISP), and covalent-soluble pectin (CSP) were measured after extraction. Briefly, 1.0 g of melon was homogenized in 10 mL of 80% (v/v) ethanol and the mixture was boiled in a water bath at 95°C for 20 min. Afterward, the mixture was cooled and centrifuged at 4,000 x g at 25°C for 10 min. The obtained residue was washed twice in 15 mL of 80% (v/v) ethanol and acetone solution to clear starch and then dissolved in 10 mL of 90% (v/v) dimethyl sulfoxide for 15 h. Finally, according to the manufacturer’s instructions, WSP, ISP, and CSP were separated and determined using the micro method .Fructose, glucose, sucrose, and starch were extracted from the melon sample as described by Fructose and sucrose were extracted as follows. 1.0 g sample from the respective treatment was homogenized in 10 mL of 80% (v/v) ethanol, and then incubated in a water bath of 80°C for 10 min. After cooling, the homogenate was centrifuged at 4,000 x g for 10 min at 25°C. 2 mg activated carbon was added to the supernatant, and decolorization was performed at 80°C for 30 min. Then 1 mL of 80% (v/v) ethanol was added, and the mixture was centrifuged at 4,000 x g for 10 min at 25°C. The supernatant was collected.-1 HClO4 was added to the residue, and extraction was performed at room temperature for 15 min. Then, 8.5 mL of double-distilled water was added and the mixture was centrifuged at 3,000 x g for 10 min at 25°C. The supernatant was diluted 4 times with distilled water for content determination.Starch extraction: about 1.0 g of the sample was homogenized in 10 mL of 80% (v/v) ethanol, and then water bathed at 80°C for 30 min. After centrifugation at 3,000 x g for 5 min at 25°C, 5 mL of double-distilled water was added to the obtained residue, which was gelatinized in boiling water for 15 min. After cooling, 3.5 mL of 9.2 mol LRespective assay kits  were used to determine the corresponding contents.-1) of PE, PG, SS, SPS, AMY, and BMY were determined using the corresponding enzyme-linked immunosorbent assay (ELISA) kits as reported by Melon samples (0.5 g) were ground in 4.5 mL 50 mM Tris-HCl buffer (pH 7.4) and the homogenate was centrifuged at 8000 x g for 15 min. The supernatant was collected for measuring enzyme activities. The activities  following the manufacturer’s instructions. The primers of selected genes were designed using the Primer Premier 5.0 software and shown in t method . GlyceraP-value < 0.05 or < 0.01 were considered significant.All statistical differences and correlation analyses were performed using SPSS 17.0  software. Data are presented as means ± standard errors; Data with The fruit firmness in both the control and chitosan-treated melons decreased throughout the storage at 3°C (P < 0.05) the weight loss after 18 d of storage, corresponding to 26.99%, 24.49%, and 24.52% decrease than that in control fruits on day 18, 24 and 30, respectively.The weight loss of melon fruit increased continuously during the 30 d of storage (As shown in P < 0.05) than in control fruits.The chilling injury appeared after 6 d of storage and then continued in both the control and chitosan-treated melons .The CSP content in melons gradually decreased during the cold storage period (P < 0.05) lower than in chitosan-treated melons at 30 d.During 30 d cold storage, the PE activity first increased to peak at 18 d, and then declined in both groups (P < 0.05) difference between chitosan-treated and control fruits. Afterward, the trend changed rapidly, and the PG activity in chitosan-treated melons turned significantly (P < 0.05) lower than in control melons, except on the day 30th.The PG activity in melons showed a fluctuating upward trend during the 30 d cold storage (LOC103482964 and LOC103499033) increased until day 18 in control fruits and day 24 in chitosan-treated fruits and then decreased. Notably, throughout the storage period, the expression of PE genes was lower in chitosan-treated fruits than in control fruits. During the 30 d cold storage, the expression of two PG genes, LOC103491984 and LOC103504264, gradually decreased to reach the minimum on the 18th and 24th d, respectively, and then increased in both groups (As shown in P < 0.05). The trends of change in fructose and glucose contents were also similar in both groups  higher than in control fruits, except on the 6th d. Correspondingly, the SS (LOC103483781) and SPS (LOC103496894) genes were upregulated in chitosan-treated melons during the 6-30 d of cold storage  along with AMY gene expression (AMY activity fluctuated in control and chitosan-treated fruits (P < 0.01). Starch and sucrose contents were positively correlated with the CSP content and negatively correlated with PG activity and WSP and ISP contents (P < 0.01) in both fruits. Softening indices showed a highly negative correlation with the glucose and fructose contents in chitosan-treated melons (P < 0.05). SS activity was positively correlated with CSP content and PE activity (0.20 < r2 < 0.52), and negatively correlated with WSP and ISP contents and PG activity (-0.62 < r2 < -0.22) in both types of fruits. SPS activity was significantly positively correlated with PE activity (P < 0.01). AMY and BMY activities were positively correlated with WSP and ISP contents in both groups; however, the correlation was more significant in chitosan-treated fruits. A highly significant positive correlation of AMY activity was observed with PE and PG activities (P < 0.05) and between BMY and PG activities in chitosan-treated fruits (P < 0.01). Altogether, correlation analysis revealed that melon fruit softening is associated with starch and sucrose metabolism, which can affect the quality of cold-stored melons.The correlations between fruit firmness, cell wall degradation parameters, and starch and sucrose metabolism indices were analyzed in chitosan-treated and control fruits Table 1.The edible coating is a modified atmosphere method that has been applied to preserve the quality of various fruit, such as tropical fruit, citrus, melon, tomato, and pome fruit . ChitosaFruit softening is a crucial parameter that determines the length of the storage period and commercial value of post-harvest fruit. In our study, chitosan treatment effectively delayed the firmness loss in melons during 30 d of cold storage Figure 2Chitosan treatment was shown to delay fruit softening in many fruits . ChitosaAccompany the melon softening, the sucrose and starch content gradually changes. Starch and sucrose metabolism in fruit is the main factor regulating soluble sugar content, which causes the change in SSC, an important sensory parameter of fruit quality. Chitosan treatment could be used to preserve higher SSC in some fruits such as longan  and oranSucrose content influences the quality, taste, and membrane stability of fruits . TherefoStarch and sucrose are also key signaling molecules in plant stress response. The relationship between chilling injury and sugar content has been confirmed in several fruits . The degChitosan treatment has been reported to reduce starch degradation in harvested fruits. LOC103483781) and SPS (LOC103496894) genes were upregulated by chitosan treatment, resulting in a higher content of sucrose in treated fruit (LOC103490827) and AMY (LOC103488899 and LOC103491451) genes were upregulated in chitosan-treated melon during the later stage of cold storage (BMY gene was shown to increase starch accumulation and decrease soluble sugar levels (BMY1 and AMY3 genes in Arabidopsis, increasing starch degradation (AMY and BMY genes.Transcriptional regulations play an important role in regulating starch and sucrose metabolism under cold stress. Cold stress increases the expression of several genes related to sucrose and starch metabolism, which improves cold stress resistance . In thised fruit . Under ced fruit . Thus, cr levels . For exaradation . SimilarIn addition to chilling injury, sucrose and starch metabolism are often related to fruit softening . We founThe correlation results revealed a certain link between fruit softening and sucrose and starch metabolism in melon during cold storage. These two complex processes involve multiple regulators including metabolites, enzyme activities, and gene regulations, which need to be investigated in future studies.Our results showed that chitosan coating effectively reduced the conversion of CSP, inhibited the increase of WSP content, and maintained fruit firmness during cold storage of melon at 3°C. Moreover, PE and PG activities were lowered along with the downregulation of related genes. Our results also concluded that chitosan treatment alleviated chilling injury in cold-stored melons by regulating starch and sucrose metabolism. In addition, a strong correlation between melon fruit softening and starch and sucrose metabolism was observed. Altogether, chitosan coating could be a reliable and potential preservation technology for mitigating chilling injury and extending the postharvest shelf life of cold-stored melons. However, further studies are needed to comprehensively elucidate the other possible mechanisms of enhance chilling tolerance after chitosan treatment.The original contributions presented in the study are included in the article/QZ: Data curation, software, formal analysis, writing - original draft, writing - review & editing. FT: Methodology, project administration. WC: Software. BP: Validation. MN: Validation. CS: Funding acquisition, supervision, visualization. XY: Investigation, conceptualization. All authors contributed to the article and approved the submitted version.


The abstracts and full texts of the resulting articles were reviewed for final inclusion. Fifty-four articles were included in this review. The analyzed topics were: (1) the composition of first- and second-generation zirconia materials (Y-TZP), (2) the behavior of the studied generations in relation to mechanical and optical properties, and (3) the modifications that were carried out on third-generation (5Y-TZP) and fourth-generation (4Y-TZP) zirconia materials. However, studies focusing on these specific characteristics in third- and fourth-generation zirconia materials are scarce. The review shows that there is a lack of sufficient knowledge about the chemical modifications of zirconia in the new generations.The aim of this study was to perform a narrative review to identify the modifications applied to the chemical structure of third- and fourth-generation zirconia ceramics and to determine the influence of these changes on the mechanical and optical properties. A bibliographical search using relevant keywords was conducted in the PubMed They transform from one phase into another, induced by a combination of temperature, humidity, and stress stimulus [The polycrystalline structure of zirconium dioxide  is a ceramic system that exhibits high strength, fracture toughness, hardness, wear resistance, good frictional and non-magnetic behavior, electrical insulation, low thermal conductivity, corrosion resistance in acids and alkalis, modulus of elasticity similar to steel, and coefficient of thermal expansion similar to iron. However, poor translucency has been one of the characteristics to improve in this material ,27,28,29Yttria-stabilized tetragonal zirconia polycrystalline is termed Y-TZP. Zirconia-based ceramics used for biomedical purposes typically exist as a metastable tetragonal partially stabilized zirconia (PSZ), which means that trapped energy still exists within the material, preventing the system from transforming into the monoclinic phase at room temperatures. However, 3 mol% yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) suffers a phase transformation when mechanical or physical stress is applied. It has been observed that fatigue creates micro-cracks in the structure of zirconia materials. When a crack propagates in 3Y-TZP , a stress-induced transformation occurs at the end of the crack and only the particles near this transform from the tetragonal into the monoclinic phase; this process is called transformation toughening (TT) ,10. This2) phase spontaneously transforms to the monoclinic (m-ZrO2) phase as a response to the variation of temperature in the mouth and the presence of humidity , regardless of any mechanical stress. The consequences of hydrothermal aging are surface roughening due to loss of crystals, enhanced wear rates, detrimental strength, and fracture toughness, followed by catastrophic failures [On the other hand, 3Y-TZP under oral conditions has shown an accelerated aging, affecting a long lasting prosthesis. Scientists who have investigated the biomaterial behavior have named this phenomenon as “low temperature degradation (LTD)”, where the tetragonal zirconia ; second-generation 3 mol% Y2O3 0.05% Al2O3; third-generation 5 mol% Y2O3 0.05% Al2O3 53% cubic structure (5Y-TZP); fourth-generation 4 mol% Y2O3 0.05% Al2O3 (4Y-TZP). To maintain stability of the crystalline form at room temperature yttria, magnesia, and other oxides have been added to zirconia materials. Güth et al.  describeIn 2015, a new ceramic system was introduced to the market: the third-generation 5-mol% yttria-stabilized tetragonal zirconia polycrystal (5Y-TZP). Increasing the yttria content (Y₃O₂) to 5 mol% was a modification that offered enhancement in the translucency of zirconia. The result is a fully stabilized zirconia with a stable cubic–tetragonal microstructure. The cubic phase reaches approximately 50% of the structure. The size and number of the crystals, which are larger than the 3Y-TZP, favor the light transmission, reducing the refraction effect and giving better translucency. On the other hand, an increase in the number of cubic crystals affects the crack propagation pattern, reducing the flexural strength and the fracture toughness of the material ,10,12,30It is relevant to recall the findings attained by Zhang et al.  in 2020 The mechanism for increasing yttria oxide as a strategy to reach a higher translucency in the zirconia materials involves better aging stability but lower flexural strength and fracture toughness, mainly due to the change in the phase composition ,37,38,39In 2017, manufacturing companies launched the fourth-generation 4-mol% yttria-stabilized tetragonal zirconia polycrystals (4Y-TZP) of zirconia. Compared with the third-generation, the yttria content was reduced to 4-mol%, which led to an enhancement of the flexural strength and fracture toughness, with a combined reduction in translucency ,10,14,31The aim of this study was to retrieve the papers that analyze the modifications performed in the chemical structure of third- and fourth-generation zirconia ceramics in order to determine how these structural changes have influenced the strength, toughness, and translucency of these materials. ® and EBSCO) and that are published in of the most influential indexed journals in the materials and dental field. A first search was performed in October 2021 and a second search was conducted in July of 2022. This study consisted of an extensive search of the literature available on dental materials worldwide. The search was limited exclusively to papers in English, between 2015 and 2022, that feature on the two main search engines , meta-analysis, systematic reviews, in vitro studies, and literature reviews were included in the present study. The following characteristics were taken into account: zirconia materials, translucent zirconia, cubic zirconia, ultra-translucent zirconia, mechanical properties, chemical structure, and load bearing of translucent zirconia.Books and documents, all papers in a foreign language (not in the English language), and zirconia materials investigated in other applications different from fixed prosthodontics were excluded.The search strategy was designed and set up by one reviewer (A.A.), who also performed the electronic search. All the studies that fulfilled the inclusion criteria were included in this review. The title and abstracts of all articles identified by the electronic search were read and assessed by one of the authors (A.A.). A shortlist of studies was then compiled and subjected to full text analysis and data extraction by the contributing authors. A manual search of the analyzed articles was also carried out. The methodological quality of all selected full-text articles was assessed using the guidelines given by PRISMA and CONSORT .First, the authors analyzed those papers that described zirconium oxide as a pure element in nature and its modifications, including its chemical structure, phase transformation, and low-temperature degradation. Second, the authors classified those papers that reported data on third- and fourth-generation zirconia materials. The original search strategy—based on keywords mentioned—resulted in 322 papers. However, the total number of papers that met the inclusion criteria for the review was 64, whereby 45.6% addressed first- and second-generation zirconia materials and 54.3% studied third- and fourth-generation zirconia materials. A total of 64 articles was reviewed, of which 20 were discarded and 44 related to third- and fourth-generation zirconia were used, with a further 10 articles added to address generalities of the review. The flow chart of the obtained results of the literature search is given in This study aimed to understand the chemical composition of zirconia ceramics and to determine which modifications have been carried out. The first selection of papers allowed the authors to categorize the composition of first- and second-generation zirconia materials (Y-TZP) and to identify the behavior of the studied generations of zirconia materials regarding abrasiveness and wear, marginal accuracy, and cementation. The second selection permitted to establish the modifications that were carried out in third-generation (5Y-TZP) and fourth-generation (4Y-TZP) zirconia materials, allowing to understand how these modifications have influenced the flexural strength, fracture toughness, and translucency of the available materials. Studies that addressed these specific characteristics in third- and fourth-generation are scarce.All selected papers are distributed according to the information they offer regarding the composition and features of zirconia materials. A summary of the in vitro studies and the observational study articles reviewed in this paper can be found in Zirconia materials have evolved into several formulations, depending on powder composition, sintering additives, heat treatment, and other processing factors.2O3 0.05% Al2O3. Due to 53% of its structure being found in cubic polycrystals, it received the name of cubic zirconia or the so-called fully stabilized zirconia (FSZ) [For Y-TZP, the translucency properties are tailored by its cubic content, which can be controlled by both the yttria content and the sintering temperature. Increasing the yttria content gives way to third-generation zirconia materials (5Y-TZP or 5Y-0.05Al) 5 mol% Yia (FSZ) ,19,35,36In general, a higher yttria content and sintering temperature will have a greater cubic content and better translucency. Nevertheless, this also triggers a lower strength and toughness ,5,10,12.2O3 0.05% Al2O3 exhibits 30% translucency, 900 MPa BFS, and better aging resistance [Fourth-generation zirconia materials are created to enhance strength and toughness, but again translucency and light reflection are diminished. The (4Y-TZP) 4 mol% Ysistance ,18,19.Currently, three zirconia grades based on the percentage of yttria content are available for monolithic dental restorations, namely 3Y-, 4Y-, and 5Y-PSZ . These three grades have a broad and inversely proportional balance between translucency and strength and, therefore, offer different clinical indications ,20,21,222O3 stabilizer was already homogeneously distributed inside, the zirconia starting powder had a significantly higher translucency, biaxial strength, and aging stability, demonstrating that the cubic content and the microstructure of the remaining tetragonal grains had considerable influence on the properties of 4Y- and 5Y-PSZ  [Zhang investigated the remaining tetragonal phase and its role in 4Y- and 5Y-PSZ . Both materials had similar basic properties. However, 5Y-PSZ  had a variation on the microstructure. When 5Y-PSZ was processed from an yttria co-precipitated powder, in which the 5 mol% Yirconia) ,25,26,27The literature supports the findings regarding the difference in the properties between third- and fourth-generation zirconia materials. The third-generation shows better optical properties than the fourth-generation zirconia materials, but the fourth-generation zirconia exhibits better strength. The abrasiveness and wear are closely related to grain size; thus, third- and fourth-generation zirconia show a similar grade of wear that is very close to enamel wear ,12,15,23Most of the studies that analyze the ageing of zirconia ceramics focus on the study of the influence of changes generated by hydrothermal conditions in the oral environment. These changes are influenced by the phase transformation of the zirconia and at the same time the affectation caused by crack propagation in the fracture, which is studied by using compressive stress. The aging resistance is higher in third- than fourth-generation zirconia due to the cubic phase percentage ,32,38,42The manufacturing and processing techniques affect the optical properties of zirconia-based restorations ,17,30,45Despite the previous efforts that have been devoted to the study of this material, there is evidently a lack of a comprehensive understanding of the chemical structure. An absence of unified terminology and several strategies adopted by manufacturing companies to improve the characteristics of this ceramic system originate a gap in the knowledge that allows mistakes when clinicians and lab technicians are using Y-TZP as a ceramic material. A limitation of this review of the literature is that the behavior of new generations of zirconia under cyclic fatigue has not been analyzed. Future research needs to analyze the cyclic fatigue and the clinical behavior of this material.Zirconia ceramics are widely used in the biomedical field. Regarding the physicochemical features and optical and mechanical properties, the expectancy of an enhancement of the mechanical properties with a combined reduction in its light optical properties when comparing the third- and fourth-generation zirconia was confirmed. The fourth-generation zirconia material 4Y-TZP shows better mechanical properties but less percentage of translucency. Clinicians should be careful when zirconia material is their choice when performing fixed restorations. Translucency is not always an advantage; if discolored stumps were the base of their restoration, translucent zirconia materials would become disadvantageous.


Starch is a naturally occurring material showing high potential for use in food packaging because of its low cost, natural abundance and high biodegradability. Over the years, different starch-based packaging films have been developed, but the impact of botanical sources on film performance has rarely been exploited. Efforts devoted to exploiting the role played by the clusteroluminescence of starch in food packaging are also lacking. This study fills these gaps by comparing the properties of edible starch films generated from different botanical sources  in food packaging. Such films are produced by solution casting. They are highly homogeneous, with a thickness of 55–65 μm. Variations in the botanical sources of starch have no significant impact on the color parameters  and morphological features of the films but affect the water vapor permeability, maximum tensile strength and elongation at break. Starch films from water chestnut show the highest percentage of transmittance, whereas those from potatoes are the opaquest. No observable change in the intensity of clusteroluminescence occurs when a packaging bag generated from starch is used to package fresh or frozen chicken breast meat; however, a remarkable decline in the intensity of luminescence is noted when the frozen meat is thawed inside the bag. Our results reveal the impact of starch sources on the performance of starch films in food packaging and demonstrate the possibility of using the clusteroluminescence of starch as an indicator to reveal the state of packaged frozen food. Micropogonias furnieri, followed by the incorporation of oregano essential oil. Such films have been found to inhibit peroxidase activity and have demonstrated the potential to be used for anti-browning packaging in fruits and vegetables [Starch is a naturally occurring material that is cheap and highly biodegradable ,2,3. Ovegetables . Recentlgetables . The abogetables ,13,14, dDespite this, starch is a complex material that presents several levels of organization ,16. It cMaize starch (MS) and water chestnut starch (WS) were purchased from Laoyao Co., Ltd.  and Yuhua Co., Ltd. , respectively. Potato starch (PS) was purchased from Yuhua Co., Ltd. . A suspension was made by dispersing 0.8 g of starch in 5 mL of distilled water. The suspension was poured, under magnetic stirring, into 15 mL of distilled water, which was preheated to 80 °C. The resulting suspension was heated additionally at 80 °C for 3 min before it was drop-casted onto the surface of a cleaned glass slide. The glass slide was then kept in vacuum at 40 °C for 10 h until complete evaporation of the solvent. Films generated from MS, WS and PS were designated as FMS, FWS and FPS, respectively.A handheld digital micrometer  with an accuracy of 0.001 mm was used to measure the thickness of a film at 20 randomly selected locations. An average of the 20 measurements was taken as the thickness of the film sample. −1 with a resolution of 4 cm−1 and reported as an average of 16 scans. The structures of MS, WS, and PS were characterized using FT-IR spectroscopy  under ambient conditions. Spectra were collected in the range of 700–3900 cm1H-NMR spectra of starch were recorded with a 400 MHz NMR spectrophotometer . DMSO-d6 was used as a solvent.−1 for 25 min. The column temperature was maintained at 35 °C. The eluent was monitored using a refractive index detector . Molecular weight determination was performed using a high-performance size exclusion chromatography (HPSEC) system  equipped with a PLGel 5 μm Mixed-C column . During analysis, 0.1 g of starch was dissolved in 10 mL of DMSO. Upon filtration through a 0.45 μm polyvinylidene fluoride (PVDF) membrane , the sample was injected and eluted with DMSO at a flow rate of 1 mL minThe amylose content of starch was determined as previously described . The exp−1.TGA and DTG of starch were performed using a Q50 TGA  equipped with platinum pans. The study was conducted in an inert atmosphere of nitrogen in a temperature range from 40 °C to 400 °C. The heating rate was set at 10 °C min−1 until breakage occurred.The tensile strength of films with a rectangular shape  was examined using a tensile tester . During analysis, the films were subjected to a strain rate of 30 mm minFilms were sputter-coated with gold. Their morphological features were observed using a JEOL JSM-6380  microscope operated at an accelerating voltage of 10 kV.⁎), redness (a⁎) and yellowness (b⁎) values of the films were obtained. The experiments were performed in triplicate.Transmittance spectra of FWS, FMS and FPS were obtained in the range of 200–800 nm under ambient conditions using an ultraviolet-visible (UV-Vis) spectrophotometer  equipped with a quartz window plate. A holder in the vertical position was used during measurement. Photoluminescence (PL) spectra of the films in both dry and hydrated states were recorded using an FLS920P fluorescence spectrometer . PL spectra were recorded at an excitation wavelength of 365 nm. The color and haze of FWS, FMS and FPS were studied using a chroma and haze meter . Film color was determined based on the CIELAB color system. The haze, lightness (L2 at 37 °C. An appropriate amount of the film was ground using a mortar and pestle and resuspended in the fresh cell culture medium to obtain a suspension with the desired concentration. The suspension was filtered using a 0.45-μm polytetrafluoroethylene (PTFE) filter . The cell culture medium in each well was replaced with 100 µL of the filtrate. After incubation at 37 °C for 5 h, the filtrate in each well was replaced with the fresh cell culture medium. The CellTiter 96 AQueous non-radioactive cell proliferation assay  was performed according to the manufacturer’s instructions to determine cell viability (%) in each well, either immediately or after 24 h of post-treatment incubation. The experiments were performed in triplicate.3T3 fibroblasts and HEK293 were cultured in DMEM supplemented with 10% fetal bovine serum. Cells were seeded in a 96-well plate 24 h before the assay at an initial density of 10,000 cells per well and incubated under a humidified atmosphere of 5% COSessile drop contact angle analysis of FWS, FMS and FPS was conducted using a video-based contact angle measurement system  incorporated with a software-controlled dosing-volume weight drop. All measurements were performed using water. The water vapor permeability of FWS, FMS and FPS was determined as previously reported . To deteMature Gala apples and boneless, skinless chicken breasts were obtained from local stores . Apples showing no wound signals were used. Each apple was cut into 12 pieces, with the weight of each piece being around 8.5 ± 0.5 g. One apple slice was put into a 50 mL centrifuge tube. A hole with a diameter of 1.5 cm was made on the cap of the tube. The hole was covered with a film affixed to the cap. One tube in which the hole was uncovered was adopted as the control. The tubes were incubated at 4 ± 1 °C. Changes in the weight of the tubes were determined at regular time intervals. The experiments were performed in triplicate.2. A meat cube was either placed directly under ambient conditions or put inside a packaging bag generated from FMS before being placed under ambient conditions. Changes in the weight of the meat cube were determined at regular time intervals. The luminescence of the bags in which meat cubes in different states  were placed was recorded using a digital camera under UV light irradiation at 365 nm. The experiments were performed in triplicate.Boneless, skinless chicken breasts were cut into rectangular pieces, with the surface area of each piece being around 20 cmp-value < 0.05 were considered to be statistically significant.All data were expressed as the means ± standard deviation. Student’s t-test was performed to assess statistical significance. Differences with a Starch is a naturally occurring polymer that exhibits blue luminescence upon excitation at 365 nm A. The me−1. This signal is attributed to stretching vibrations of the hydroxyl groups of starch and water, as previously described [−1 is attributed to C-H stretching vibrations. The bands at 1147 and 1046 cm−1 are assigned to C-O stretching vibrations of the glucose ring. The peak at 991 cm−1 is due to C-O stretching vibrations of the C-O-C bond in the α-1,6-linkage.Despite variations in the amylose content, the NMR spectra of WS, MS and PS show the same pattern C, with cescribed . The peaThe thermal properties of starch samples and the corresponding films were determined using TGA and DTG E,F. The Solution casting was adopted to generate edible films from WS, MS and PS. The generated films are highly homogeneous, with thickness ranging from 55 to 65 μm A,B. The Opacity is an important property of food packaging films because it, on one hand, affects the degree of consumer acceptance and, on the other hand, may contribute to protection of sensitive food components from degradation by absorbing or reflecting a fraction of the incident light to slow down degradation reactions . AlthougFor a film to be used as a direct food contact material during food packaging, it must demonstrate a lack of toxicity . The toxThe generated starch films are used to produce packaging bags, which exhibit blue luminescence upon excitation at 365 nm due to the process of CTE, for food packaging A. VariatTo demonstrate the performance of the films in protecting packaged food from moisture loss, apple pieces are adopted as a food model A,B. The Starch exhibits high biodegradability and natural abundance, showing the potential to overcome some of the problems  caused by synthetic plastics in food packaging. In this study, we compare the properties of edible starch films generated from different botanical sources  in food packaging. All tested films are optically transparent in the visible range (400–700 nm), with the percentage of transmittance ranging from 50% to 80%. Variations in the botanical sources of starch lead to changes in diverse film properties , although the impact on color parameters and morphological features of the films is not apparent. The ability of our films to reduce moisture loss of packaged food was confirmed by using apple slices and chicken breast meat as food models. In addition, by taking advantage of the moisture-mediated change in the intensity of clusteroluminescence exhibited by the generated films, we demonstrated the potential of the edible starch film to reveal whether packaged frozen meat has undergone freeze–thaw cycles during storage and transportation. Not only do our results reveal the impact of starch sources on the performance of starch films in food packaging; they also provide a new perspective on the use of starch films in food packaging, extending the application potential of such films from mere food protection as demonstrated in conventional studies ,52,53 to


Starch is a very abundant and renewable carbohydrate and an important feedstock for industrial applications. However, most starch-based products are not cost-efficient due to the high energy input needed in traditional enzymatic starch conversion processes. Raw-starch-digesting enzymes (RSDEs) from filamentous fungi have great commercial value in starch processing. However, the regulatory mechanisms associated with their production in filamentous fungi remain unknown.Penicillium oxalicum 114-2 has broad RSDE activity. Four regulators, including the amylase transcription activator AmyR, the catabolite repression repressor CreA, the group III G protein α subunit PGA3, and the nonhistone chromosomal protein HepA, have been found to play a crucial regulatory role in RSDE expression. Enzymatic assays revealed that RSDE production significantly increased after the overexpression of AmyR and HepA, the deletion of CreA and the dominant activation of PGA3. RT-qPCR analysis demonstrated that there is a mutual regulation mode between the four regulators, and then formed a cascade regulation mechanism that is involved in RSDE expression. Comparative transcriptomic analysis between the wild-type strain and genetically engineered strains revealed differentially expressed genes that may mediate the RSDE expression.In this study, we reported the novel finding that cellulolytic fungus The four different types of regulators were systematically investigated and found to form a regulatory network controlling RSDE gene expression. Our results provide a new insight into the regulatory mechanism of fungal amylolytic enzyme expression and offer a theoretical basis to rationally improve the RSDE yield in the future.The online version contains supplementary material available at 10.1186/s13068-022-02162-6. Starch is the major carbohydrate reserve polymer in a large variety of higher plants, such as maize, cassava, wheat, potato, and oat. It is a potential substrate for the production of sugars and liquid fuels and chemicals –3. Due tAspergillus sp. and Rhizopus sp. and Bacillus sp. are the most widely used and possess the following advantages: low production cost, good consistency, small time and space required for production, and easy process modification and optimization [Pontibacillus sp. ZY, and the AmyZ1 could efficiently hydrolyse raw starches at a relatively low temperature [P. oxalicum, was heterologously expressed by P. pastoris, displaying a high capacity for raw starch degradation and remarkable stability [Aspergillus species [P. oxalicum HP7-1 in the presence of glucose and starch revealed five transcription factors exhibiting significant regulation in the production of RSDEs. Among them, a transcription factor containing a pair of SANT/Myb domains controls the expression of an important RSDE gene, POX01356/PoxGA15A, in P. oxalicum [. In addition to these regulators, the HMG-box protein PoxHmbB and the extracellular protease activator PrtT, were identified as RSDE repressors in P. oxalicum [. However, their research is insufficient to elucidate the regulatory mechanism behind the RSDE gene expression. Therefore, to further reduce the cost of enzyme production, it is rational to breed fungal strains based on an understanding of the molecular mechanisms controlling RSDE gene expression.To reduce the energy costs associated with traditional starch biorefineries, raw-starch-digesting enzymes (RSDEs) could directly degrade raw starch granules into oligosaccharides or glucose below the gelatinization temperature of starch . It has mization . Althougmization , 10–13. perature . Additiotability . On the tability , rationa species , 17. Recoxalicum . In addioxalicum , 20. HowP. oxalicum exhibits good prospects for raw amylase production and application in recent years [P. oxalicum 114-2 has received substantial attention due to its highly efficient apparatus for cellulolytic enzyme synthesis. It has been under investigation for more than 30 years in China [P. oxalicum. However, secretome analysis performed through LC–MS/MS showed that Amy15A and Amy13A, which occupied 28.85% and 10.95% of the total protein, respectively, were the two major amylolytic enzymes in the wild-type strain [P. oxalicum114-2 over the years [. Regulators, such as the classic TFs AmyR and CreA, nonclassical TFs, including a casein kinase CK2, proteins heterotrimeric G protein, and nonhistone chromosomal protein, have been identified to be involved in cellulase expression pathways [P. oxalicum. By employing RT-qPCR and RNA-seq technologies, the regulatory network was revealed and provided deep insights into the RSDE expression mode of P. oxalicum. Additionally, the suggested mechanisms behind regulators of RSDE expression might be general properties in amylolytic fungi and broadly enable engineering strategies for the protein hyperproducers.Apparently, nt years , 15, 18.in China . Accordie strain , 22. Numhe years . Regulatpathways –26. AlthPontibacillus sp. and P. oxalicum GXU20 [To analyse the raw starch-digesting abilities of extracellular protein from WT 114-2, the strain was cultivated in Vogel’s minimal medium with starch as a sole carbon source and amylolytic enzyme expression inducer. Soluble starch and different raw starches derived from rice, corn, potato, buckwheat, and cassava were used as reaction substrates. The extracellular amylases showed different raw starch-digesting enzyme activities. Compared with soluble starch as a substrate, the extracellular enzymes exhibited higher RSDE activity towards raw rice starch, but lower RSDE activity towards the other four raw starches . Figure amyR overexpressed under the ubiD strong constitutive promoter), ΔCreA (deletion of the creA gene), and Mpga3 (expressing a dominantly activated PGA3 with substitution of Gln208 by leucine), and OhepA (hepA overexpressed under the ubiD strong constitutive promoter). In particular, HepA was first identified for its role in regulating amylolytic enzyme expression in P. oxalicum. The four transformants were purified and identified by Southern blot and gene sequencing  while restricting mycelium extension in nutrient limiting conditions (starch medium). Spores of the OhepA strain germinated and grew slower on PDA, while a similar phenotype on starch was observed compared with the WT strain , were selected for evaluation in WT 114-2 and four engineered strains. As shown in Fig. amyR on rice starch was upregulated to different extents in the four engineered strains relative to the WT , PDE_06088 (Zn_clus) and PDE_09536  were found to be co-upregulated, while only PDE_02772 (Zn_clus) was found to be co-downregulated in the four engineered strains.Generally, glycoside hydrolase expression is regulated by upstream transcription factor(s) in filamentous fungi , 23, 28.P. oxalicum 114-2 on different raw starches. The results showed that the amylolytic enzymes secreted by the 114-2 strain had comprehensive degradation activity towards raw starch from different sources. The RSDEs from P. oxalicum 114-2 showed better amylolytic activity against rice raw starch, while they exhibited a faster deconstruction rate when degrading starch granules  may be partially due to enhanced AmyR expression  leads to GDP–GTP exchange on the Gα subunit and its activation , 33. Theion Fig. A. Taken ion Fig. . We furtWhile RSDE-assisted starch industrial processing has been a topic of study over the years, the gaps in our knowledge regarding the RSDE expression and the regulation of the pathways could be considered actual bottlenecks in the development of more effective genetic engineering strategies. In this study, we identified a number of common genes and metabolic pathways that mediate RSDE expression through global transcriptome analysis of four engineered strains. These common upregulated or downregulated genes are most likely to be involved in the regulation of RSDE expression, which will provide reduced but valuable targets for the next step of optimizing the regulatory network. These sophisticated regulatory mechanisms may also function to optimize carbon and energy flux. We will further investigate these common genes to explore more signalling transmitters or regulatory factors to improve the regulatory network of RSDE expression, thus providing theories for the most fruitful means of genetic modification to obtain higher industrial RSDE yield in the future.7 per mL were inoculated into and grown in liquid glucose minimal medium for 24 h. Then, mycelia were collected by vacuum filtration, and 0.5 g wet mycelia were resuspended in 100 mL liquid VMM supplemented with 1% (wt/vol) soluble starch for further growth. The cultures were maintained at 30 °C on a rotary shaker at 200 rpm. The fermentation supernatant was used for microscopic observation of starch degradation and amylolytic enzyme activity determination.The wild-type strain 114-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC 5302 and used throughout this work as a control. Raw starches  were purchased from a local market in Changsha, China. The raw starch substrates used for the enzymatic activity assay were washed at least three times with ultra-pure water. Soluble starch was purchased from Sigma-Aldrich . The strains listed in Table hph gene and DNA fragments approximately 2.0 kb upstream and downstream of the target gene, and the DNA fragments were amplified by PCR using the corresponding primer pairs , which contains a mutant site, was synthesized. The template of the pga3 mutagenesis cassette was obtained by fusing the Mpga3 coding region, hph cassette, and 3' flanking region .To determine the integration type of targeting cassettes in transformants, Southern blot analyses were performed , and complementary DNA was generated using a reverse transcription kit following the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Premix Ex Taq™   with the primers listed in Additional file P. oxalicum 114-2 genome. Transcript abundance  genes with significantly different expression levels were identified through a significance test with combined thresholds (FDR ≤ 0.01 and fold change ≥ 2). Pearson’s correlation coefficient was used to evaluate transcriptome reliability, and three biological replicates were used in each sample. Differential gene expression was analysed by using the Majorbio cloud computing platform, included a series of DESeq software packages.Fresh spores of each strain were inoculated into 100 mL of 1% glucose medium and incubated at 200 rpm and 30 °C for 24 h. Equal amounts of mycelia were transferred to 1% raw rice starch medium and cultured for 21 h. Total RNA was extracted from the different samples by using a TRIzol total RNA extraction kit  and treated with DNase I  to remove the DNA. Transcriptome assays based on Illumina sequencing technology were performed at Shanghai Majorbio Biopharm Technology Co., Ltd . After quality control, the generated clean reads were mapped against predicted transcripts from the Additional file 1: Fig. S1. Southern blot analysis of the genomic DNA of OamyR, OhepA, ΔCreA, and Mpga3 mutants. The location of the probes and restriction enzyme sites for Southern blot analysis are shown. The primers are listed in Table S2. (A) The overexpression strains OamyR and OhepA generated 5.6 and 4.7 kb DNA bands, respectively, while the parental strain 114–2 did not produce a detectable band, indicating that the overexpression cassettes were integrated into the genome. (B) Southern blot analysis of the genomic DNA of 114-2 and Mpga3. The 114-2 strain generated a 1.3 kb DNA band, while a 3.1 kb band was obtained in Mpga3 indicating that the targeted gene was correctly replaced. Sequencing of the mutated pga3 (Mpga3) gene in strain Mpga3. The mutated codon is indicated. (C) Southern blot analysis of the genomic DNA of 114-2 and ΔCreA. The 114-2 strain generated a 3.3 kb DNA band, while a 6.3 kb band was obtained in ΔCreA indicating that the targeted gene was correctly replaced. Fig. S2. RSDE activity assay of WT and various mutants. The strains were cultivated in liquid VMM supplemented with 1% starch and cultivated at 30 °C for 5 days. Soluble starch was used as a reaction substrate for RSDE activity assays. Fig. S3. RSDE activity assay of WT, ΔHepA and ΔPGA3 strain. The strains were cultivated in liquid VMM supplemented with 1% starch and cultivated at 30 °C for 5 days. Raw rice starch was used as a reaction substrate for RSDE activity assays. Fig. S4. Extracellular proteins of 114-2 on starch analysed by SDS-PAGE. 32 μL culture supernatant of 114-2 strain was loaded after cultivating in 1% starch medium for 72 h, respectively. Lane M was the molecular weight marker, lanes 1 and 2 represent two independent cultivations for 114-2 strain. Fig. S5. SDS-PAGE analysis of the purified raw starch digesting enzyme Amy15A. Lane M indicates protein molecular weight marker; lane 1 indicates the purified Amy15A. Fig. S6. Effects of pH and temperature on enzymatic activity and the stability of the RSDE Amy15A. (A) The effect of pH on enzyme activity. The enzyme activity was assayed in a citrate–phosphate buffer (pH 3.0–7.0) at 37 °C. (B) The pH stability of Amy15A was measured by pre-incubating the enzyme in various buffers for 24 h at 4 °C, and the residual enzyme activity was determined using the standard method. (C) The influence of temperature on enzyme activity. The enzyme activity was determined between 35 °C and 80 °C under optimum pH condition. (D) The influence of temperature on enzyme stability. Temperature stability was determined by the standard method after pre-incubating the enzyme at pH 4.5 between 30 °C and 75 °C for 1 h. Data given are mean ± standard deviation from three replicates. The results are from a representative experiment, and similar results were obtained in two other independent experiments. Table S1. Quality control statistics. Table S2. Primers used in this study.


International Journal of Neonatal Screening was able to uphold its high standards for published papers due to the outstanding efforts of our reviewers. Thanks to the efforts of our reviewers in 2022, the median time to first decision was 21 days and the median time to publication was 49 days. Regardless of whether the articles they examined were ultimately published, the editors would like to express their appreciation and thank the following reviewers for the time and dedication that they have shown International Journal of Neonatal Screening:Aishwarya, SiddharthMarcão, AnaAlcántara-Ortigoza, Miguel AngelMessina, Maria AnnaAnderson, SharonMillington, David S.Argudo Ramírez, AnaMincarone, PierpaoloArmstrong, Kate L.Moat, StuartAsamoah, AlexanderMonostori, PéterBakkeheim, EgilMunck, AnneBasheeruddin, KhajaNakamura-Utsunomiya, AkariBhattacharjee, ArindamOrsini, JoeBonham, JimPalasanthiran, PamelaBorrajo, Gustavo J. C.Palma, SilviaBrockow, InkenParad, Richard B.Bush, Lynn WeinPesch, MeganCabello, Juan FranciscoPitt, JamesCarling, RachelPng, May EeCocho, José A.Purcell, Patricia L.Cordovado, Suzanne KehoeRegelmann, Molly O.Coughlin, Curtis R.Ribes, AntoniaCunningham, AmyRizzo, CristianoDangouloff, TamaraRizzo, WilliamDe Hora, Mark R.Rocha, HugoDe Winter-de Groot, Karin M.Rock, Michael J.Dekkers, Eugenie H. B. M.Ruoppolo, MargheritaDhiman, VineetScharfe, CurtDluholucký, SvetozárSchulze, AndreasEyskens, FrancoisSciortino, StanleyFarrell, Philip M.Servais, LaurentFicicioglu, CanShone, ScottFingerhut, RalphSicko, Robert J.Franková, VěraSomech, RazGaviglio, AmyStepien, Karolina M.Gibson, James B.Tajima, GoGoldenberg, AaronTang, HaoGrosse, Scott D.Tangeraas, TrineHall, KateTavakoli, Norma P.Hall, PatriciaTaylor, JenniferHeather, NatashaTerlizzi, VitoHinton, Cynthia F.Tewari, Vishal VishnuHwu, Wuh-LiangTherrell, BradfordIshige, NobuyukiTluczek, AudreyJalali, AliVan Vliet, GuyJones, KendraVeraguth, DorotheKemper, Alex R.Vockley, Cate WalshKimizu, TomokazuVon Döbeln, UlrikaKingsmore, Stephen F.Wang, RaymondKucera, Katerina S.Webster, DianneLilian, DownieWedell, AnnaLund, Allan MeldgaardWiberley-Bradford, Amy E.Mak, Chloe MiuWiley, VeronicaMallack, EricYahyaoui, RaquelManning, AdrienneZetterström, RolfHigh-quality academic publishing is built on rigorous peer review.


Correction to: Journal of Clinical Immunology10.1007/s10875-021-01177-2Due to typesetting mistake, the published PDF and HTML versions did not match.The original version has been corrected.


This study was aimed at preparing zirconia samples via additive manufacturing (AM) and subtractive manufacturing (SM) and testing the following aspects: (1) the manufacturing accuracy of the zirconia samples and (2) the bond strength of porcelain to zirconia to evaluate the applicability of the zirconia fabricated by AM in dental clinics. We used three milling machines for SM  and a 3D printer for AM (AO). The manufacturing accuracy of the zirconia specimen in the internal and marginal areas was evaluated by superimposing techniques to calculate the root mean square (RMS) values. The bond strengths of porcelain to zirconia prepared via SM and AM were measured using a universal testing machine. The internal and marginal RMS values of the zirconia prepared by AM (AO) were within the range of those of the zirconia prepared by SM . Moreover, the bond strength value of the zirconia prepared by AM (35.12 ± 4.09 MPa) was significantly higher than that of the zirconia prepared by SM (30.26 ± 5.20 MPa). Therefore, AM technology has significant potential for applications in dentistry. Digital technology has recently been incorporated into analog-based dental technology. New methods for manufacturing digital dental products have been developed since the introduction of computer-aided design (CAD)/computer-aided manufacturing (CAM) systems in dentistry . CAD/CAMAccording to the manufacturing methods, CAD/CAM systems are classified as subtractive manufacturing (SM) and additive manufacturing (AM) techniques. SM is widely used to prepare digital dental products, such as zirconia prostheses, with complicated conventional manufacturing methods ,4. SM teNumerous AM technologies have been proposed recently in dentistry, such as stereolithography, fused deposition modeling, and selective laser sintering (SLS), since the introduction of 3D model production technology by Kodama in 1981 ,8,9,10. Zirconia has been known as one of the representative ceramic biomaterials since it was first proposed for use in the medical field in 1969 . ZirconiIn addition, for zirconia manufactured with the AM technology to be used in dental practice, the zirconia-based dental prosthesis must have micro-level accuracy to ensure a perfect fit with the abutment and prevent secondary caries in the oral cavity. Several studies have evaluated the accuracy of zirconia dental prostheses manufactured with SM technology ,23,24. MThe objective of this study was to estimate the manufacturing accuracy of zirconia dental products prepared via the AM and SM technologies. In addition, we compared the bond strength of porcelain to zirconia produced via the AM and SM technologies. We also examined the feasibility of using AM technology for producing zirconia prostheses for use in dentistry.We used three milling machines for SM and one digital light processing (DLP)-type 3D printer for AM to prepare the zirconia specimens. Furthermore, we used a zirconia blank for SM and a zirconia slurry for AM. Detailed information on the devices and materials used in this study is listed in RMS) values [RMS value:Xi1, is the i-th point in the CRD, Xi2, is the i-th point in the CTD, and n denotes the total number of points measured for RMS analysis. The marginal area accuracy was evaluated by separately extracting 1.5 mm from the marginal end in the image of the zirconia specimen along the inner direction, as shown in To evaluate the accuracy of the zirconia specimens prepared by AM and SM, we superimposed the CTD and CRD using 3D image analysis software . We then calculated the distance between the points on the two image surfaces as root mean square  values ,26. The To assess the bond strength of porcelain to zirconia that was prepared by AM and SM, we used the debonding/crack-initiation test specified in the International Standard, “ISO 9693-2 Dentistry―Compatibility testing―Part 2: Ceramic-ceramic systems” . We usedThe accuracy and bond strength test values were expressed as mean ± standard deviation. We analyzed the RMS values of the accuracy using one-way analysis of variance and the Games–Howell test as a post-hoc test . We also analyzed the bond strength results using an independent t-test. All statistical analyses were performed at a significance level of 95% (α = 0.05).p < 0.05). Moreover, the RMS evaluation of the marginal area demonstrated superior fitting accuracy in the following order: AR (21.06 ± 11.71 μm), K5 (25.20 ± 9.38 μm), AO (48.75 ± 4.39 μm), and UP (48.93 ± 4.90 μm). The RMS values of the AR and K5 groups were significantly lower than those of the AO and UP groups (p < 0.05).p < 0.05).The SEM results obtained by observing the debonded surface indicated the formation of more dimples on the surface of the AM-fabricated zirconia specimens , which wIn this study, we aimed to evaluate the feasibility of using zirconia prepared via AM technology in dental practice. To this end, we assessed the manufacturing accuracy of the dental prostheses completed with the final sintering. Most studies that examined the machining accuracy evaluated the accuracy of the results after machining processes that did not include the sintering process; thus, they were based on purely machined samples. However, the sintering process, which compensates for approximately 20~30% of the thermal shrinkage, is required in general to obtain the final dental product in SM technology. Therefore, this study, which evaluated the manufacturing accuracy using specimens completed with final sintering, fills a gap in the literature.The results of the RMS values of the zirconia specimens prepared via AM and SM indicated that the AR and UP groups, which were SM groups, showed the best and worst accuracy in the internal  and marginal areas among all experimental groups, respectively. Moreover, the accuracy of the AO group (AM group) was within the range of accuracy of the three SM groups. During the manufacturing process using the milling machines (SM devices), several factors, such as the directions of the tool paths, type of milling conditions and burrs, and the software that was used for CAM, influenced the deviation of the accuracy of the dental products ,29,30. IAM also includes several variables that affect the dimensional accuracy of the final product in the digital CAD/CAM systems during scanning, design, fabrication, and sintering . Among tp < 0.05). In this test, we failed to directly fabricate a zirconia specimen for the three-point flexural strength test using both the AM and SM methods. As the test specimen was significantly thin (0.5 mm), fracture and deformation occurred during the manufacturing process; thus, we prepared large zirconia blocks in advance and then cut them into the final test specimen. This fabrication procedure excluded the effect of the difference in the surface roughness of the AM- and SM-fabricated zirconia specimens because we used the same cutting device and polishing process. The bond strength test results indicated that the strength value of the AM group was higher than that of the SM group.The bond strength test showed that the bond strength in the AM zirconia group was significantly higher than that in the SM zirconia group  . As the Within the limitations of this study, the manufacturing accuracy of the zirconia specimen produced by the AM device was within the range of that of the zirconia specimen produced by three different SM devices. In addition, the bond strength value of porcelain to zirconia produced by AM technology was higher than that in the specimen fabricated via SM technology. Furthermore, SEM observation confirmed that a higher number dimples—which implies a strong bond between porcelain and zirconia—were found in the AM zirconia specimen. Therefore, we hypothesize that a dental prosthesis based on AM technology has a considerably high potential for use in dental clinics, and additional research is required for its practical application in dentistry.


Behçet's disease is a multisystem vasculitis whose pathogenesis remains unclear. Although usually described in young adults, it may begin in childhood. The diagnosis is clinical, based on international criteria. The limitations of early diagnosis are related to the progressive onset of symptoms and the variety of differential diagnoses at this age in the absence of a pathognomonic diagnostic test.To report the epidemiological features of our series and to compare the 2015 pediatric criteria with the 2014 and 1990 international criteria.This is a monocentric, retrospective study of 31 children over an 11-year period from January 2011 to December 2021. All patients suspected with Behcet's disease by a pediatric rheumatologist were included in the study, the 2014 international criteria “ISG 2014” being the gold standard classification criteria used in our center.p = 0.002) compared with the 2014 international criteria taken as gold standard, as well as the 1990 international criteria with a respective sensitivity and specificity of 52.6% and 91.7% (p = 0.034).31 cases of Behcet's were collected. The mean age was 10 years (5.5–16) ± 2.87 years. A female predominance is noted with a sex ratio F/M of 1.21. A quarter of the patients were from a consanguineous marriage and 38.7% had a family history of Behcet disease. Mucocutaneous manifestations were represented by recurrent oral aphthosis 87.1%, genital aphthosis 29%, pseudofolliculitis 48.4%, erythema nodosum 6.5% and acne lesions 3.2%. The pathergy test was positive in 1 case. Ocular involvement was reported in 29% and joint involvement in 45.2%. Thromboembolic complication was seen in 9.6% and neurobehcet in one patient. The HLA B51 antigen was present in 45.2% of cases. In our series 38.7% respond the pediatric criteria, while 61.3% met the 2014 international criteria and 35.5% met the 1990 international criteria. The 2015 pediatric criteria have respectively a sensitivity and specificity of 63.2% and 100% (p = 0.002).Our study highlights a female predominance, a high rate of consanguinity and familial Behcet. The sensitivity and specificity of the 1990 pediatric and international criteria appear to be better than those of 2014, with a more significant trend for the 2015 pediatric criteria (


Acinetobacter spp. have emerged as troublesome pathogens due to their multi-drug resistance. The majority of the work to date has focused on the antibiotic resistance profile of Acinetobacter baumannii. Although A. calcoaceticus strains are isolated in the hospital setting, limited information is available on these closely related species. Methods & Results: The computational analysis of antibiotic resistance genes in 1441 Acinetobacter genomes revealed that A. calcoaceticus harbored a similar repertoire of multi-drug efflux pump and beta-lactam resistance genes as A. baumannii, leading us to speculate that A. calcoaceticus would have a similar antibiotic resistance profile to A. baumannii. To profile the resistance patterns of A. calcoaceticus, strains were examined by Kirby–Bauer disk diffusion and phenotypic microarrays. We found that Acinetobacter strains were moderately to highly resistant to certain antibiotics within fluoroquinolones, aminoglycosides, tetracyclines, and other antibiotic classes. These data indicate that A. calcoaceticus has a similar antibiotic resistance profile as A. baumannii ATCC 19606. We also identified that all Acinetobacter species were sensitive to 5-fluoroorotic acid, novobiocin, and benzethonium chloride. Conclusion: Collectively, these data provide new insights into the antibiotic resistance in A. calcoaceticus and identify several antibiotics that could be beneficial in treating Acinetobacter infections.Background:  Acinetobacter spp. are Gram-negative bacteria that have become a pressing health concern over the past few decades. Worldwide, Acinetobacter spp. make up 20% of the infections seen in the hospital setting and are considered the main cause of hospital-acquired bacteremia and pneumonia [Acinetobacter spp. are characterized by their intrinsic antimicrobial resistance [Acinetobacter strains are increasingly resistant to the major classes of antimicrobials, including beta-lactams, aminoglycosides, and fluoroquinolones [Acinetobacter isolates has increased in recent years [Acinetobacter are among the most serious MDR organisms [neumonia . Once ininolones . Due to inolones . Unfortunt years , and, acrganisms .Acinetobacter baumannii-calcoaceticus (ABC) complex is a group of phenotypically indistinguishable opportunistic pathogens that cause hospital-acquired bacteremia and pneumonia. Among the ABC complex members, A. baumannii, A. pittii, and A. nosocomialis are the most commonly isolated strains [A. calcoaceticus strains are also isolated, but less frequently [Acinetobacter have focused on A. baumannii [A. baumannii strains are resistant to most first-line antibiotics. Several studies have examined the antibiotic resistance in ABC complex isolates but have not distinguished between species [A. calcoaceticus, we may be able to identify antibiotics to which multiple species are sensitive. Currently, few studies have specifically examined the resistance profile of A. calcoaceticus strains. Herein, we sought to address this gap in knowledge by examining the antibiotic sensitivity profile of commercially available A. calcoaceticus and clinical isolates of A. calcoaceticus.The  strains ,7. A. caequently . Based oaumannii . These s species ,10. DeteAcinetobacter species are well documented for their ability to counteract the effects of antibiotics through multidrug efflux pumps and targeted mechanisms. Since antibiotic resistance mechanisms are commonly studied in A. baumannii species, we sought to compare the genomic capacity of A. calcoaceticus strains to resist antibiotics to that of A. baumannii and unclassified Acinetobacter strains. We identified 1290 genomes of A. baumannii, 23 genomes of A. calcoaceticus, and 128 genomes of unclassified Acinetobacter spp. in the Integrated Microbial Genomes (IMG) database  and examined key genes in the antibiotic resistance pathways, as identified in the KEGG database   production, beta-lactamases, penicillin-binding proteins (PBPs), and the increased activity of efflux pumps [A. baumannii, A. calcoaceticus, and Acinetobacter spp. genomes did not have genes encoding the beta-lactam transporters OprD, OmpF, OmpC, OmpU, or PorB, indicating that these bacteria can limit the uptake of beta-lactams into the outer membrane. We also found that >95% of the A. baumannii and A. calcoaceticus genomes harbored Class C (AmpC) beta-lactamases. Some A. baumannii genomes contained the genes for Class A , Class D , and Class B  beta-lactamases. Only a few A. calcoaceticus genomes had Class A (4.3%) beta-lactamases, and no genomes had Class B or D beta-lactamases. Interestingly, only 21% of unclassified Acinetobacter species harbored Class C beta-lactamases, and 8.6% had Class C beta-lactamases. These findings suggest that A. calcoaceticus is similar to A. baumannii in terms of its genomic capacity to resist antibiotics.Despite the diverse targets of antibiotic classes A, Acineter 2021) B. We fouux pumps . In our A. calcoaceticus, we examined commercially available A. calcoaceticus ATCC 23055 and A. calcoaceticus CB1 and A. calcoaceticus clinical isolates  using the Kirby Bauer Disc Diffusion Assay  and colistin (miscellaneous antibiotic). All strains had an intermediate resistance to ceftazidime , meropenem (carbapenem), and tetracycline (tetracycline) and were sensitive to imipenem (carbapenem), amikacin (aminoglycoside), gentamicin (aminoglycoside), tobramycin (aminoglycoside), and tigecycline (tetracycline) (To examine the antibiotic resistance profile of on Assay . For comcycline) .Acinetobacter strains were moderately resistant to the cephalosporins and sensitive to the carbapenem imipenem. We also examined fluoroquinolones and found that all the strains were moderately resistant to the antibiotics lomefloxacin, enoxacin, and ofloxacin  complexes where the specific species cannot be distinguished. In our study, we found several antibiotics that significantly reduced the growth of Acinetobacter spp. in drug classifications that are not normally used to treat these organisms. For example, we found that Acinetobacter spp. are sensitive to novobiocin. Novobiocin interferes with the ATPase activity of DNA gyrase and inhibits the DNA repair/replication processes initiated by double-strand breaks [A. baumannii strain ATCC 17978 [A. baumannii AB5075 [A. calcoaceticus strains and an unclassified Acinetobacter species. These data suggest that novobiocin could serve as a potential therapy for patients with Acinetobacter infections. Additionally, combination drugs can be explored, such as novobiocin and rifampicin. Recently, nine dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) were found to potentiate novobiocin and rifampicin activity against multidrug-resistant (MDR) clinical isolates of A. baumannii [To date, there have only been a few studies that directly focus on efazolin . To the d breaks . ImportaCC 17978  and be ei AB5075 . Our stuaumannii .Acinetobacter strains were highly sensitive to 5-fluoroorotic acid. 5-fluoroorotic acid has been used experimentally for many years in yeasts and archaea studies [A. calcoaceticus, unclassified Acinetobacter spp. ATCC 27244, and A. baumannii ATCC 19606 are highly sensitive to 5-fluoroorotic acid. This finding is consistent with a previous study that found that the A. baumannii strains DR2 and AB067 were also sensitive to 5-fluoroorotic acid [We also found that all  studies . Non-toxtic acid . Importatic acid , indicatA. calcoaceticus strains.A major limitation of our study is that we did not identify minimum inhibitor concentrations (MICs) for the strains. Additionally, our Biolog Phenotypic Microarrays were performed in a chemically defined medium. The advantage of using a chemically defined medium is that the supernatant is amenable to downstream LC-MS/MS analysis since it does not contain components such as peptone and yeast extract. We have used this defined medium to examine the growth of diverse microbes and measure bacterial metabolites and proteins by LC-MS/MS ,21,22,23A. calcoaceticus strains respond to antibiotics and demonstrates that A. calcoaceticus behaves in a similar manner as A. baumannii in terms of antibiotic resistance. We believe this work can provide new insights into Acinetobacter resistance and new targets for antibiotic therapy.This work sheds light onto how A.calcoaceticus ATCC 23055 and A. calcoaceticus Carolina Biological (CB1) strains were purchased for use in this study. Four clinical isolates of A. calcoaceticus  were obtained [Acinetobacter baumannii ATCC 19606 and Acinetobacter spp. ATCC 27244 were used. All strains were cultured on Leeds Acinetobacter agar, a selective and differential medium for Acinetobacter spp. [600nm) on a Spectronic 200 spectrophotometer .ter spp.  Single cAcinetobacter strains was determined using the Kirby–Bauer method. Antibiotic disks were purchased from FisherScientific  and include: 30 µg Amikacin (cat# CT0107B); 30 µg Ceftazidime (cat# CT0412B); 10 µg Gentamicin (cat# CT0024B); 10 µg Colistin (cat# CT0017B); 15 µg Erythocycline (cat# 237093); 10 µg Imipenem (cat# CT0455B); 10 µg Meropenem (cat# CT0774B); 30 µg Tetracycline (cat# 230998); 15 µg Tigecycline (cat# CT1841B); and 10 µg Tobramycin (cat# CT0056B). Bacteria were diluted at a McFarland of 0.5 and incubated on Mueller Hinton Agar plates overnight (20 h) at 37 °C with the antibiotic disks. Zone diameters were recorded, and the susceptibility results were categorized as sensitive, moderately resistant, or resistant based on the CLSI breakpoint criteria [The antibiotic sensitivity of criteria .Acinetobacter in the presence of antibiotics, overnight BHI cultures were adjusted to an OD600nm = 0.1 in chemically defined media termed ZMB1 [600nm adjusted ZMB1 was placed into each 96 well of the PM11 and PM12B Phenotype Microarray Biolog plate . Growth was monitored after 16 h of incubation at 37 °C on a Synergy HT BioTek plate reader. Antibiotic-treated microbes were compared to untreated microbes in ZMB1, and strains were considered to be sensitive to an antibiotic when there was <50% growth with an applied antibiotic compared to untreated microbes. Strains were considered moderately resistant to an antibiotic when there was 25–50% growth and highly resistant to an antibiotic when there was >75% growth in antibiotics compared to growth without antibiotics.To measure the growth of med ZMB1 . A totalAcinetobacter genomes were identified in the Integrated Microbial Genomes (IMG) database (img.jgi.doe.gov) available through the Joint Genomes Institute (JGI)  [A. baumannii, 23 genomes of A. calcoaceticus, and 128 genomes of unclassified Acinetobacter spp. The genes involved in beta-lactam resistance were identified in the KEGG database . Based on a KEGG pathway analysis, we queried the Acinetobacter genomes for the following antibiotic-resistant genes: TolC (K12340), AcrA (K03585), AcrB (K18138), MexA (K03585), MexB (K18138), OrpM (K18139), RND (K18146), MFP (K18145), OMP (K12340), AdeA (K18145), AdeB (K18146), AdeC (K18147), blaTEM , Bla2 , AmpC , Pxa23 , OprD (K18093), OmpF (K09476), OmpC (K09475), OmpU (K08720), PorB (K18133), PBP1a/2 (K05366), PBP2 (K05515), and Fts1 (K03587).CA, USA) . We idenp < 0.05 value was considered significant.The data are presented as the mean ± standard deviation. Comparisons between the groups were made with One-way Analysis of Variance (ANOVA). GraphPad was used to generate the graphs and statistics . A *


Anacardium occidentale L.) is a commercially important plant. Cashew nuts are a popular food source that belong to the tree nut family. Tree nuts are one of the eight major food allergens identified by the Food and Drug Administration in the USA. Allergies to cashew nuts cause severe and systemic immune reactions. Tree nut allergies are frequently fatal and are becoming more common. Aim: We aimed to identify the key allergenic epitopes of cashew nut proteins by correlating the phage display epitope prediction results with bioinformatics analysis. Design: We predicted and experimentally confirmed cashew nut allergen antigenic peptides, which we named Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily). The Ana o 2 and Ana o 3 epitopes were predicted using DNAstar and PyMoL (incorporated in the Swiss-model package). The predicted weak and strong epitopes were synthesized as peptides. The related phage library was built. The peptides were also tested using phage display technology. The expressed antigens were tested and confirmed using microtiter plates coated with pooled human sera from patients with cashew nut allergies or healthy controls. Results: The Ana o 2 epitopes were represented by four linear peptides, with the epitopes corresponding to amino acids 108–111, 113–119, 181–186, and 218–224. Furthermore, the identified Ana o 3 epitopes corresponding to amino acids 10–24, 13–27, 39–49, 66–70, 101–106, 107–114, and 115–122 were also screened out and chosen as the key allergenic epitopes. Discussion: The Ana o 3 epitopes accounted for more than 40% of the total amino acid sequence of the protein; thus, Ana o 3 is potentially more allergenic than Ana o 2. Conclusions: The bioinformatic epitope prediction produced subpar results in this study. Furthermore, the phage display method was extremely effective in identifying the allergenic epitopes of cashew nut proteins. The key allergenic epitopes were chosen, providing important information for the study of cashew nut allergens.Background: Cashew ( Bioinformatic analyses are frequently used to predict protein properties related to antigenicity. We used two antigenic proteins present in cashew nuts to compare the results of the bioinformatics-based antigenicity predictions with those of the experimental epitope mapping. Several amino acid oligopeptides, representing the predicted and unpredicted epitopes, were expressed using phage display and screened with the pooled human sera of patients with a cashew nut allergy. The findings revealed that the computer predictions did not correlate very well with the presence of linear epitopes.Anacardium occidentale L.) is a member of the Anacardiaceae family, which includes nine Anacardium species [The cashew plant ; the results are summarized in The antigen index predicts the likelihood of a protein containing epitopes. As shown in E. coli host DH5alpha. After the insert length was confirmed  of the pooled samples was much greater than the 0.35 KUA/L in the patient group and less than 0.35 KUA/L in the healthy control group (0.08). All of this resulted in the important epitopes for the allergic reaction that we are studying being IgE−binding epitopes rather than others−binding epitopes.Phage display technology was used in this study to determine the main allergen epitopes, primarily because the traditional enzyme−linked immunosorbent assay would inactivate all of the proteins and viruses, resulting in more nonspecific binding reactions of antigen epitopes and interfering with the experimental results. The phage display technology has a higher activity and less nonspecific binding. Because the synthetic peptide’s molecular weight was less than 100 bp, many PCR experiments failed, and the single-strand hybridization complementary method was finally chosen to form double strands . When buFrom the study of the bioinformatic analysis of the Ana o 2 (A) and Ana o 3 (B) features, between 180 and 195 nm, the changes in the α−helix, β−turn, and random coil were relatively stable. The α−helix decreased from 30.70% to 12.20%, the β−turn increased from 17.70% to 22.40%, and the random coil increased from 32.80% to 54.00%, with an overall trend of a decreasing α−helix and an increasing β−turn and random coil. Because of their bulge structures, the β−turn and random coil easily bind to antibodies to form epitopes, whereas the α−helix is not easily deformed and does not easily bind to antibodies to form epitopes, such as Tropomyosin, a coiled−coil protein with an α−helix structure that becomes a major allergen from shellfish. Furthermore, a high frequency of β−turn and random coil occurrence was found in the entire amino acid sequence of Ana o 2 and Ana o 3, indicating a high possibility of epitope formation in this region.A previous study used immunoblotting and 15 aa long peptides  to perfoTherefore, the fraction of aa producing allergen epitopes was 5.5%. Ana o 3 had a much larger fraction because 56 aa produced at least six and possibly seven epitopes, whereas the entire protein was only 138 aa long, resulting in an epitope−producing aa fraction of 40.6%. Ana o 3 is thought to be more likely to cause an allergic reaction than Ana o 2 due to the higher number of identified epitopes and a much smaller size.The protein was resolved using Omni PAGE precast gel (4–15%). The protein samples were prepared by mixing with 5X loading buffer (4:1 by volume) and heating for 5 min, followed by cooling at room temperature. The cashew allergen protein was purified using glucan G−150 column chromatography . SDS−PAGAfter people with a cashew allergy eat cashews, a special substance (human IgE antibody) appears in their blood, causing hypersensitivity . This st4HCO3) were added and incubated at 37 °C in a water bath for 1 h. After that, it was filled with 20 µL of 55 mM indole–3–acetic acid (IAA) (dissolved in 25 mM NH4HCO3) and left in the dark for 30 min. The samples were then fully mixed and incubated overnight at 37 °C with 0.2 µg of trypsin. The samples were centrifuged the next day and then desalted using the C18 Zip Tip column. The protein solutions were then centrifuged, vacuum freeze–dried, and kept at −20 °C.Protein samples must be pretreated before the enzymatic hydrolysis of protein solutions can take place. To begin, we took some protein samples and mixed them with 20 µL of 2 M urea. After 30 min, 20 µL of 10 mM dithiothreitol (DTT)  and the NCBI–Anacardium occidentale database. Maxquant  was also used to analyze the proteomic data.Following the mass spectrometry sequencing, the peptide sequencing data were compared to those found in the UniProt-First, the background was measured without any objects being placed in the sample pool. Following that, the sample solution was injected into the reader’s cuvette . Meanwhile, the samples were placed in a cuvette and detected three times at a 0.5 nm bandwidth, 1.0 nm step, 180–260 nm wavelength, and 0.5 s time-per-point.https://www.ncbi.nlm.nih.gov/protein/AAN76862.1?report=genpept, accessed on 8 April 2021). Then, the secondary structures of the predicted allergenic proteins were compared using the Protean program in DNAstar, with the structure and binding of the proteins determined using the CD.The allergen sequences obtained by the mass spectrometry sequencing were compared with those obtained from the NCBI database , along with predictions of their hydrophilicity, flexibility, and surface accessibility. Jin Wei Zhi Biological Co., Ltd. synthesized the DNA oligonucleotides of the predicted epitopes, with upstream and downstream primers designed to contain the XmaI and KpnI restriction sites , which added 33 bp to the length of each peptide nucleotide sequence [The aa sequences of allergens Ana o 2 and Ana o 3 were extracted from the NCBI database . The antigenic epitopes of these two proteins were predicted using the default settings of the DNAstar and PyMoL software, both of which are part of the Swiss model . The plasmid DNA was digested with the restriction enzymes KpnI and XmaI (NEB), separated on a 1.5% agarose gel, and purified using a gel recovery kit (Promega). The synthesized primers were used separately to amplify the entire predicted peptide in a reaction volume of 10 µL using Taq polymerase. The PCR conditions underwent pre-denaturation for 1 min and 20 cycles of 1 min at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by a 4 min terminal extension at 72 °C. After two cycles, the two complementary primers were combined and annealed for 1 h at 37 °C. The amplicons were separated on 5% agarose gels before being cut out, purified, and ligated into the pCANTAB5E plasmid DNA. The ligation mixture was transformed into competent E. coli DH5α cells, and the DNA extracted from single colonies was sequenced by a third party (Sangon Biotech) [The pCANTAB5E phage display system was used to express the predicted epitopes. To see if the peptides were correctly cloned into the plasmid, the epitopes were cloned and expressed in Biotech) .The recombinant plasmids were then isolated, quantified, and electroporated into TGI competent cells; the transformed cells were diluted with super optimal broth with a catabolite repression (SOC) culture medium and cultured overnight at 37 °C in 2× YT/Amp/Glu plates with 2% glucose and 100 µg/mL. The colonies on the plates were sequenced the following day.600 of 0.4–0.6 was obtained. To these exponentially growing cells, the helper phage M13KO7 (NEB) was added at a ratio of 10 or 20:1 (phage to bacteria) and incubated at 37 °C for 30 min. Following centrifugation, the cell pellets were resuspended in 150 µL of 2XTY/Amp/Kan. Because the expression of the recombinant peptides depends on the osmotic expression of the lacZ promoter with glucose removed in this step, the bacteria were cultured overnight at 37 °C and 300 rpm. Following centrifugation, 50 µL of the supernatant obtained from each well were used for the phage enzyme-linked immunosorbent assay (ELISA).A bacterial suspension (50 µL) of each primary library was added to a 96-well plate together with a 200 µL 2XYT/AMP/Glu culture medium and incubated at 37 °C overnight with a 250 rpm rotation. The cultures were stored at −70 °C with an equal volume of 100% glycerol. The subcultures were grown in 96-well plates until an ODThe plate was coated with a mouse anti-human IgE monoclonal antibody (purchased from Jackson immunoResearch Laboratories Inc.) and incubated overnight at 4 °C. The coated test plates were washed three times with PBST the next day. These plates and an uncoated control plate were blocked for 45 min in PBS with 2% BSA. After washing, the pooled human sera derived from ten individuals with a cashew allergy and the pooled human sera from the control group were added into the wells (100 µL/well) and incubated for 1 h (PBST). After washing the plates, each phage supernatant was used as the antigen (mixed 1:1 with 1% MPBS) and added into the wells. Finally, a 1:4000 dilution of monoclonal mouse anti-M13-HRP (MBL) was used as the secondary antibody. After the final wash, the substrate solutions were added to the plates for 15 min of incubation. After adding the stop solution, the plates were read on a Gen5 plate reader .Epitopes were identified on two proteins derived from cashew nuts by using phage library expression and screening with human sera obtained from patients with a cashew nut allergy. The bioinformatic predictions correlated poorly with the experimentally verified epitopes. Of the identified epitopes, Ana o 3 is more likely to cause allergic reactions than Ana o 2. Therefore, further research should focus on changing the composition or structure of the identified protein domains to produce low-allergen nuts. We conclude that cashew nut allergen epitopes can be expressed, and their physicochemical properties can be studied, by using the bacteriophage display technology, which provides a theoretical basis for allergen studies in the future.


Forty years ago, on April 28th, 1982, ESOT was founded in Zürich, Switzerland by an assembly of 14 European transplant surgeons, to satisfy the “need for a society … which would represent the aims and needs of transplantation surgery and surgeons in Europe” . As a result of the strong commitment of the ESOT community, the editorial board released the first issue containing 10 articles in 1988  and the team of Thomas Wekerle and Rainer Oberbauer (2015–2021) in their roles as editors-in-chief. The current leading editorial board is grateful to have inherited such a high-quality journal from our forebears.The ESOT founders understood the need for the society to establish a scientific journal and Gauke Kootstra was appointed as the first editor-in-chief of  in 1988 . Since tTransplant International was committed to modernity. As written in the editorial of the first issue: “We are convinced that with such modern means of communication as the fax, it is possible to keep the processing and publication times in our journal to a minimum. This will be of great importance, especially in the field of transplantation which is, by its nature, a very dynamic science”  rubies to ESOT!Traditionally, 40 years mark a ruby anniversary. As


Microstructural evolution and dynamic recrystallization (DRX) behaviors of a Ni-Cr-Mo alloy were researched utilizing hot compressive experiments. The changed features of dislocation, subgrain and grain structure correlating to forming parameters were examined by transmission electron microscope (TEM) and electron backscatter diffraction (EBSD). Results illustrate that the consumption of dislocation and the coarsening of substructure/DRX grain are prominently enhanced with an increased forming temperature. However, the annihilation/interaction of dislocation and the expansion of subgrain/DRX grain boundary can be limited at a larger strain rate. Meanwhile, considering the discrepancy in DRX variation rates concerning the strain rate’s ranges, an improved DRX kinetic model was developed. Compared to the classical DRX kinetic model, the good consistency between the forecasted and tested results demonstrates that the established improved DRX kinetic model can precisely characterize the DRX features of the Ni-Cr-Mo alloy over a wide strain rate range. Additionally, the EBSD’s quantitative statistical results proved that the variation of DRX grain size can be supremely defined as the power formulation of the forming temperature and strain rate. In hot forming, sophisticated microstructural changes often occur and markedly affect the deformation characteristics of alloys ,4,5,6,7.In past, the kinetic behaviors and microstructural changes of alloys during DRX were widely investigated ,21,22. FDue to the outstanding resistance of oxidation and corrosion, Ni-Cr-Mo alloys are extensively applied in the nuclear industry ,38,39. RIn the present work, the changes and interactions of substructure/ DRX grain structure of a Ni-Cr-Mo alloy in hot compression are studied. The variation of DRX kinetic with forming parameters is investigated. An improved dynamic recrystallization kinetic model is established, and its forecasted precision is analyzed. −1, according to the standard of GB/T 9327.4-1988. The Gleeble-3500 simulator is produced by the DSI company. The total height reductions in the tested specimens were set as 60%. The concrete step of hot formation can be formulated as follows. Every tested specimen was firstly heated to a forming temperature at 10 °C/s and maintained for 300 s. Then, the hot forming of each tested specimen was conducted, respectively. The compressed samples were swiftly cooled by water after hot compression. To investigate the changes of dislocations and substructures, 3CH2OH + 20 mL HClO4). As illustrated in In the present investigation, the utilized material is a commercial Ni-Cr-Mo alloy, and its chemical composition (wt. %) is shown in ehaviors ,9. Then, 1150 °C b. SimultNormally, the values of peak strain ( follows :(1)εp Normally, the Zener–Hollumon ( follows :(2)Z =Substituting the tested true stress-true strain results into Equation (2), the value of is 0.995 , suggestGenerally, the classical ollowing : (3)XdCommonly, for the classical method to determine  follows ,32:(4)As illustrated in Equation (4), the values of ferences . For σrus works . Commonly, the values of ified as :(6)εc The correlation of Substituting the values of According to the tested data , the meaFurthermore, substituting the values of From the above analysis, the DRX kinetics equation can be summarized as follows.T or low T or a reduction in −1 can be reasonably accepted. As −1, the discrepancy between the forecasted −1 , the evolution of Z at the strain rate ranges of 0.1–10 s−1 and 0.001–0.1 s−1 are exhibited in With respect to the tested results, the variations of Z at the strain rate ranges of 0.1–10 s–1 and 0.001–0.1 s−1 is displayed in Correspondingly, the change of −1 and 0.001–0.1 s −1 can be determined as 1.512 and 1.61, respectively.Substituting the tested Based on To verify the improved DRX kinetic model (Equation 11)), the comparisons between the assessed values and tested values of 1), the c−1 is estimated at 8.5 µm. When T, which induces the enhancement of grain boundary migration. Furthermore, the changes of DRX grain are notably influenced by −1 to 1 s−1, the lowering DRX degree can be seen, and the growth of DRX grains is limited drWe take a logarithm of Equation (12) and readjust it as follows.By substituting the experimental values of Therefore, To validate the DRX grain size predicted model (Equation (14)), the comparisons between the forecasted The microstructural changes and DRX behaviors of a Ni-Cr-Mo alloy in hot compression are researched. An improved DRX kinetic model was established to calculate the DRX features of the Ni-Cr-Mo alloy. Several significant results are summarized as follows.(1)The variations of substructures are closely correlated to forming parameters. The nucleation and interaction of dislocations can be intensified, while the refinement of subgrains/DRX grains is easily limited at high temperatures or low strain rates.(2)An improved DRX kinetic model that considers the variation characteristics of DRX behavior in the segmented ranges of strain rate is proposed. Good consistency between the forecasted and tested results demonstrates that the established model can strictly elaborate the DRX kinetic features of the researched alloy.(3)The variation of DRX grains is abruptly affected by forming parameters. At a large strain rate or low temperature, the DRX grain is distinctly refined. The mean size of DRX grains in hot forming is well described as the equation of the forming temperature and strain rate.