When the substitution of TBBF was increased from 0 to 35%, the cooking and textural properties decreased significantly (p < 0.05), while the content of bioactive compounds  increased significantly (p < 0.05). In addition, the substitution of TBBF decreased the starch digestibility of TBDNs. A 10.4% reduction in eGI values was observed in the TBDNs with 35% TBBF substitution compared to the control sample. The results of differential scanning calorimetry showed that with the increase of TBBF, TBDNs starch became more resistant to thermal processing. Meanwhile, the X-ray diffraction and Fourier transform infrared spectroscopy results revealed that the long- and short-range ordered structures of TBDN starch increased significantly (p < 0.05). Furthermore, the substitution of TBBF decreased the fluorescence intensity of α-amylase and amyloglucosidase. This study suggests that replacing TBF with TBBF could produce low glycemic index and nutrient-rich TBDNs.This study aimed to investigate the impact of partial replacement of Tartary buckwheat flour (TBF) with Tartary buckwheat bran flour (TBBF) on the quality, bioactive compounds content, and  Noodles, one of the traditional staple foods in China, are becoming popular all over the world, among which, dried noodles are becoming more and more popular in the staple food market, due to their advantages, such as the convenience of consumption, easy storage, and relatively low price . In receTartary buckwheat is rich in bioactive compounds, such as phenolic, flavonoids, and dietary fiber . As is wTartary buckwheat bran is the outermost layer of buckwheat seeds, containing more bioactive compounds compared to Tartary buckwheat flour (TBF) . RachmanThere are adverse effects of Tartary buckwheat bran on the quality of noodles, including reduced texture properties and sensory acceptability . Gluten in vitro starch digestibility of TBDNs. The study especially focused on the mechanism by which TBBF affected the digestion of TBDNs. It was hypothesized that TBBF would influence the starch digestibility of TBDNs in two ways: (i) the bioactive compounds  in the TBBF might interact with starch, affecting the structure of starch molecules; (ii) the inhibition of digestive enzymes by TBBF. To the best of our knowledge, this is the first time the effect of TBBF on the digestibility of TBDNs through the evolution of starch structure and inhibition of digestive enzymes has been studied. To verify our hypotheses, the thermal properties, crystallinity (long-range), and short-range ordered structure of TBDN starch were investigated. Meanwhile, the fluorescence quenching of α-amylase and amyloglucosidase was also studied.Thus, the aim of the present study was to investigate the influence of TBBF on the quality, bioactive compound content, and Aspergillus niger 110 U/mg), pepsin , and pancreatin  were purchased from Sigma-Aldrich Chemical Co. . A Total Dietary Fiber kit was purchased from Megazyme International Ireland Ltd. . All chemical reagents used were of analytical grade.Tartary buckwheat flour (TBF)  and Tartary buckwheat bran flour (TBBF)  were purchased from Liangshan Jianmao Food Co., Ltd. . Wheat flour  was obtained from COFCO Flour (Haining) Co., Ltd. . Wheat gluten  was obtained from Binzhou Zhongyu Food Co., Ltd. . The α-amylase , amyloglucosidase . In addition, TBBF was used to substitute TBF in the ratios of 0%, 5%, 15%, 25% and 35%, respectively. To form a dough sheet, the blended (100 g) flour and wheat gluten (7 g) were mixed evenly in a mixer  for 6 min. Then, distilled water (37.5 mL) was added and mixed for 5 min. To distribute the moisture evenly, the dough was sealed and rested in a plastic bag . After that, the dough was sheeted using a laboratory semi-automatic noodle machine . The roller gap was gradually decreased from 2.2 mm to 0.8 mm with a reduction of 0.2 mm (dough sheets were pressed three times at each roller gap), and then the dough sheets were cut into strips with a width of 2.0 mm. Finally, the noodles were dried in a laboratory intelligent drying platform . The drying procedure was gradually changed from low temperature and high humidity  to relatively high temperature and low humidity , until the moisture content of noodles reached 12%.As seen in Cooking properties, including water absorption and cooking loss, were characterized using the AACC method 66-50 . BrieflyThe textural properties of TBDNs were characterized using a TA-XT2i Texture Analyzer  based on the method described by Zou et al. . Three cv/v/v) was used to extract the phenolic and flavonoids. The mixture was shaken for 3 h in a water bath . Then, the supernatant was removed, and another 20 mL of acidified methanol was added for repeat extraction. After that, the extracts were combined and centrifuged at 4000× g for 15 min. The supernatant was collected and placed in a round bottom flask for evaporation. Finally, the supernatant was diluted to 10 mL with the same acidified methanol for further use.According to Biney et al. , freeze-2CO3 solution  was added to neutralize the reaction. Finally, the reaction solution was placed in an incubator . The absorbance of samples was determined at 760 nm using a Microplate Reader . The standard curve was made using the gallic acid, and the TPC was denoted by the gallic acid equivalents (mg GAE/g DW).The total phenolic content (TPC) of TBDNs was measured according to the study of Dulf et al. . First, 2  and AlCl3  were added to the solution, respectively, and incubated . Then NaOH  solution was used to create the alkaline environment required for the reaction. Then the reaction solution was diluted to 10 mL with deionized water, shaken well, and incubated at 25 °C for 30 min. The absorbance of samples was determined at 510 nm using a Microplate Reader . The standard curve was constructed using rutin, and the TFC was denoted by rutin equivalents (mg RE/g DW).According to the method of aluminum chloride colorimetric assay , the supDietary fiber content, including total (TDF), insoluble (IDF), and soluble dietary fiber (SDF) of TBDNs were measured by the enzyme gravimetric method using a Megazyme Dietary Fiber kit, according to the AOAC method 991.43 .in vitro simulation digestion procedure was divided into oral, gastric, and intestinal. HCl, NaOH, and various salt solutions were used to create the digestive environments corresponding to the three phases. First, TBDNs were cooked for the optimal cooking time and cut into a width of 2 mm, and accurately weighed to 5 g into a centrifuge tube (50 mL). Then, 4 mL of simulated salivary fluid (SSF) was added and vortexed thoroughly. After that, the NaOH  solution was used to adjust the pH to 7. Deionized water (955 μL) with dissolved α-amylase and CaCl2  solution were used to bring the volume of the suspensions to 10 mL. The activity of α-amylase in the suspensions from the oral phase was 750 U. Second, after shaking for 5 min (200 r/min), deionized water (1.995 mL), CaCl2  solution, and 8 mL of the simulated gastric fluids (SGF) with dissolved pepsin were added. The activity of pepsin in the suspensions from the gastric phase was 40,000 U. Third, after shaking , the NaOH  solution was used to adjust the pH to 7 for the pancreatic enzyme reaction. Bile salt (2.5 mL), CaCl2  solution, and 16 mL of simulated intestinal fluid (SIF) were mixed with the suspensions from the gastric phase. Then, the mixture was diluted to 40 mL with deionized water. The pancreatic enzyme content was determined by the activity of amylase in the suspension (40 mL), with an activity of 8000 U. Finally, the suspension was continuously shaken (200 r/min), and 0.1 mL aliquots were transferred for measurement after 0, 10, 20, 30, 60, 90, 120, 180, 240, and 300 min. To stop the reaction, 0.4 mL of anhydrous ethanol was added. Before centrifugation , the mixture was rested for 40 min. A glucose assay kit  was used to measure the glucose content, which was converted into the amount of digested starch. Furthermore, the starch digestion rate was calculated as the total starch content divided by the amount of digested starch. According to Fan et al. , the in The starch hydrolysis curves of TBDNs were made based on a nonlinear first-order rate equation:t represents the starch hydrolysis rate at time t; parameter C∞ represents the predicted maximum hydrolysis rate; and parameter k represents the starch hydrolysis constant. As described by Zou et al. [t/dt), and t was used to analyze the hydrolysis rates of different fractions of starch. Equation (2) was given, as follows:Parameter Cu et al. , noodlesThe parameter k is used to indicate the rate of starch hydrolysis. The AUC (area under hydrolysis curve), HI (hydrolysis index), eGI (estimated glycemic index), and RDS (rapidly digested starch is the amount of starch digested after 20 min), SDS (slowly digested starch is the amount of starch digested between 20 min and 120 min), RS (resistant starch is the amount of starch remaining after 120 min) were calculated using the method of Sun et al. .The thermal properties of TBDNs were analyzed using a DSC , according to the method of Han et al. . First, The XRD patterns of starch in TBDNs were determined using an X-Ray diffractometer  with Cu-Kα radiation (λ = 0.15406 nm), according to Han et al. . The exp−1 were scanned 32 times with a resolution of 4 cm−1. An empty cell was recorded as a background. In addition, the IR absorption intensity ratios at 1050 to 1022 cm−1 (R-1047/1022) and 1022 to 992 cm−1 (R-1022/992) were obtained using the method described by Liu et al. [−1 were baseline corrected, smoothed, and deconvoluted using omnic software (version 8.0) with the half-width and enhancement factors of 19 cm−1 and 2.1. The spectra of starch in TBDNs were determined using a FTIR spectrometer . First, freeze-dried TBDNs flour was mixed with KBr in a proportion of 1:150. Then the mixture was pressed into transparent sheets, and loaded onto the ATR cell. After that, the spectra of 4000–400 cmg for 15 min. After that, the supernatant was transferred for the assay. Samples (1 mL) were scanned in the range of 300–400 nm, with a scanning rate of 1200 nm/min. The excitation wavelength, excitation slit width, and emission slit width were 288 nm, 5 nm, and 5 nm, respectively.The quenching effect of TBDNs on α-amylase and amyloglucosidase was measured using a fluorescence spectrometer  according to Liu et al. . Freeze-p < 0.05) using SPSS 26. LOS and non-linear fitting of starch hydrolysis rate were performed using origin 2021.All data are presented as means ± standard deviations. The statistical significance was analyzed using one-way ANOVA followed by Duncan’s test , from 5.86 and 5.36 for the control to 12.32 and 7.95 for the noodles with 35% TBBF substitution (TBBF-35), respectively. Compared with raw noodles, both the TPC and TFC of cooked noodles decreased. Surprisingly, total phenolic and flavonoids retentions were highest in the noodles with 10% TBBF substitution (TBBF-10) and 15% TBBF substitution (TBBF-15), respectively. This was consistent with the results of the TBDN hardness. This was possibly because the dietary fiber filled in the gluten network, which helped encapsulate the bioactive compounds and protected them from thermal degradation. According to Fu et al. [As seen in u et al. , TBBF cou et al. . The lowp < 0.05).In this study, the content of TDF, IDF, and SDF were determined and are presented in in vitro digestion curves of starch decreased with increasing levels of TBBF substitution (p < 0.05) decreased from 54.59% of the control to 50.73% with TBBF-35 when the digestion proceeded to 300 min. ∞) decreased significantly (p < 0.05), but k increased slightly when the substitution with TBBF reached 35%. Moreover, with increased addition of TBBF, the RDS and SDS dropped from 16.78% and 27.93% to 15.29% and 23.20%, respectively, while the RS increased significantly (p < 0.05), from 55.29% to 61.51%. These results suggest that the TBDNs starch was difficult to digest, due to the substitution of TBBF. This might have been because TBBF contains more dietary fiber and polyphenols, which reduced the digestibility of the TBDNs [t/dt)), and that reveals how the digestion rate changed during the digestion process [in vitro digestion curves of the starch were fit with the LOS. As seen in 1 and k2 are the digestion rate constants for the two different starch fractions. 1 decreased slightly and then increased, while k2 significantly (p < 0.05) decreased from 1.50 to 0.61. The decrease of k1 and k2 indicates that the starch of TBDNs were more difficult to digest. In addition, the increase of k1 with the 35% TBBF substitution could have been due to the severe disruption of the gluten network, resulting in an increased exposure of starch to digestive enzymes [p < 0.05) increased  of TBDNs significantly increased, from 63.37 °C to 67.70 °C (p < 0.05), while the peak (Tp) and conclusion temperature (Tc) changed slightly. In addition, the enthalpy change of gelatinization (ΔH) gradually decreased from 3.66 J/g to 1.81 J/g. The increase of To was due to the dietary fiber hindering starch from contacting the water, which limited the swelling and gelatinization of starch [Thermal characteristics of the TBDNs are shown in f starch . In addif starch . As showf starch ,45. In af starch  reportedp < 0.05) increased, from 20.15 (Control) to 24.55 (TBBF-35), an increase of 22%. The reason for this might have been that the dietary fiber competed with starch for water absorption, resulting in less contact of the starch with water and protecting the structural integrity of the starch. In addition, the dietary fiber interacted with the crystalline region of amylopectin, due to its polyhydroxy structure, which increased the density of double helix accumulation [The XRD results of all TBDNs are presented in mulation . Moreovemulation .−1 represents the O–H stretching vibration, the absorption peak at 2930 cm−1 is related to the C–H stretching vibration, and the characteristic peaks at 1200–800 cm−1 in the spectrum were mainly caused by the vibrations of starch polysaccharides, which could reflect the conformation of starch [−1 is associated with the double helix structure of the starch crystalline region, the absorption peak at around 1022 cm−1 corresponds to the amorphous region of starch, and the absorption peak at around 992 cm−1 represents hydrogen bonding [The FTIR spectra of Tartary buckwheat noodles with different TBBF substitutions are given in f starch . In part bonding . As obse bonding ,52.R-1050/1022 and R-1022/992  were useTo further understand the mechanism by which TBBF reduced the starch digestibility, α-amylase and amyloglucosidase fluorescence quenching of different TBBF substitutions for TBDNs were studied. As shown in in vitro starch digestibility of TBDNs. Compared to the control sample, the C∞ and eGI values of TBBF-35 were significantly (p < 0.05) reduced, by 5.79% and 6.32, respectively. The LOS fitting results showed that TBBF mainly influenced the starch digestion rate of the latter part. From the evolution of the starch structure of the TBDNs, the increase in long- and short-range ordered structures resulted in more resistance to thermal processing and enzymatic digestion of TBDN starch. In addition, the fluorescence quenching intensity was enhanced as the proportion of TBBF increased. Thus, the substitution of TBBF resulted in an ordered starch structure and low enzyme activity, which reduced the starch digestibility of TBDNs. Although the addition of TBBF decreased the cooking properties, tensile strength, and elasticity of TBDNs, it increased the content of bioactive compounds and the textural attributes were still acceptable with a proper substitution of TBBF. This study proved that TBBF can be used as an ingredient to decrease the GI value of noodles.The replacement of TBF with TBBF decreased the


Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing Mycoplasma pneumonia of swine (MPS), brings massive economic losses worldwide. Genomic variability and post-translational protein modification can enhance the immune evasion of Mhp, which makes MPS prone to recurrent outbreaks on farms, even with vaccination or other treatments. The reverse vaccinology pipeline has been developed as an attractive potential method for vaccine development due to its high efficiency and applicability. In this study, a multi-epitope vaccine for Mhp was developed, and its immune responses were evaluated in mice and piglets. Genomic core proteins of Mhp were retrieved through pan-genome analysis, and four immunodominant antigens were screened by host homologous protein removal, membrane protein screening, and virulence factor identification. One immunodominant antigen, AAV27984.1 (membrane nuclease), was expressed by E. coli and named rMhp597. For epitope prioritization, 35 B-cell-derived epitopes were identified from the four immunodominant antigens, and 10 MHC-I and 6 MHC-II binding epitopes were further identified. The MHC-I/II binding epitopes were merged and combined to produce recombinant proteins MhpMEV and MhpMEVC6His, which were used for animal immunization and structural analysis, respectively. Immunization of mice and piglets demonstrated that MhpMEV could induce humoral and cellular immune responses. The mouse serum antibodies could detect all 11 synthetic epitopes, and the piglet antiserum suppressed the nuclease activity of rMhp597. Moreover, piglet serum antibodies could also detect cultured Mhp strain 168. In summary, this study provides immunoassay results for a multi-epitope vaccine derived from the reverse vaccinology pipeline, and offers an alternative vaccine for MPS. Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing MPS [sing MPS , brings sing MPS . Commercsing MPS , and Mhpsing MPS . We propsing MPS . As showsing MPS ,7,8 and sing MPS , which lWith the development of computational immunology and vaccine informatics, a reverse vaccinology pipeline has been established , and somIn this study, we developed a multi-epitope vaccine for MPS, MhpMEV, based on the reverse vaccinology pipeline. The secondary structure of MhpMEV was determined by circular dichroism, and the three-dimensional structure was predicted and validated in silico. Moreover, the immunogenicity of MhpMEV was evaluated in mice and piglets, indicating that it could induce humoral and cellular immune responses. For pan-genome analysis, Roary was installed in a Bio-Linux 8 system, and the dependency check is shown in ABCPred found 312 potential B-cell epitopes with default parameters within the four immunodominant antigens, and Bcepred screened 282 linear B-cell epitopes based on physical and chemical properties. Then, the BLASTp program was performed to find the epitopes that were co-identified by the two services. Finally, 35 non-redundant B-cell epitopes were identified, each 16 amino acids in length. The screening processes are presented in EpiTOP 3.0 was used to search for MHC-II binding epitopes within the 35 non-redundant B-cell epitopes and 11 epitopes were identified that could bind to more than three MHC II alleles. The antigenicity of the 11 epitopes was calculated by VaxiJen V2.0, and epitopes DSEIQKLKKPELNSLE and TFFLDGKSSFSKSDNK were removed, as their antigenic potential was less than 0.5. Through the prediction of MHCPred server version 2.0, 58 sub-epitopes were screened that could bind to predominant HLA-II allele DRB1*0101 (IC50 < 500 nM), and the length of these epitopes was split into nine amino acids. Then, the 58 epitopes were submitted to VirulentPred to determine their cytotoxicity, and all of them were positive. IFNepitope predicted the ability to induce IFN-γ production and identified 10 epitopes, which were regarded as MHC-II binding epitopes. The results of the selection process of MHC-II binding epitopes are shown in The MHC-I binding prediction tool v2.23 found 43 potential epitopes within the four immunodominant antigens, which could bind to porcine MHC-I alleles with IC50 < 500 nM. The length of each epitope was split into nine amino acids, but some epitopes contained overlapping sequences. Then, their immunogenicity was calculated by the MHC I immunogenicity score, and 22 candidate epitopes with a score >0 were selected. To further improve the accuracy, the obtained epitopes were submitted to MHCPred server version 2.0 to calculate their binding efficiency with the predominant HLA-A*0201, and eight potential epitopes were identified. Finally, VaxiJen V2.0 prioritized the MHC-I binding epitopes and identified six epitopes as “possible antigens”, which were regarded as MHC-I binding epitopes. The results of the selection process of MHC-I binding epitopes are shown in E. coli BL21(DE3) competent cells for protein expression. The open reading frame features and MhpMEV protein sequence are presented in MHC-I/II binding epitopes that contained overlapping sequences were merged and finally 11 non-redundant MHC-I/II binding epitopes were obtained. These epitopes are shown in Four induction conditions were explored for MhpMEV expression, and the results are shown in MhpMEV was redesigned as MhpMEVC6His for further purification, which added a 6×His tag to its C-terminus directly . The secRobetta, I-TASSER, and AlphaFold were used to predict the three-dimensional structure of MhpMEVC6His, and the results are shown in The molecular model of MhpMEVC6His predicted by Robetta was docked with swine immune-associated receptors through ZDOCK, including MHC class I antigens  and Toll-like receptor 3 (2A0Z). The top 10 binding models with the highest ZDOCK scores for each receptor are listed in p = 0.2240; 42 d, p = 0.0015) and IgG1 levels  between MhpMEV-25 and MhpMEV-50 groups. However, at 42 d, serum antibodies from the MhpMEV-25 group showed higher levels against rMhp597 and the 11 synthetic epitopes, except for QVDGYFIGL (p < 0.0001), when compared with the MhpMEV-50 group. Moreover, the serum antibodies from the Mhp group could detect all of the synthetic epitopes compared to the PBS group at 42 d (p < 0.0001). The results of the lymphocyte proliferation assay are shown in p < 0.0001; 42 d, p < 0.0001). Between the MhpMEV-25 and MhpMEV-50 groups, IFN-γ levels  and splenic lymphocyte proliferation showed no significant differences , but MhpMEV-50 induced higher levels of IL-4 at 35 and 42 d . Mouse immunization was performed to evaluate the immunogenicity of MhpMEV, with Freund’s adjuvant used as the immunostimulant. The vaccination schedule of the mouse immune experiment is presented in p < 0.0001). The results of the IFN-γ assay are presented in p < 0.0001), but these two groups did not show significant differences. We also determined the serum antibody titer against rMhp597 at 28 d, and the titer was between 1000 and 1500; the result is presented in The vaccination schedule of the piglet immune experiment is presented in Streptococcus pneumoniae [Klebsiella pneumoniae), which indicates that the pipeline for candidate antigen selection is strict [In the co-evolution of pathogens and host immune systems, pathogens with immune evasion mechanisms have been reported, including influenza A virus (IAV) , SARS-Coeumoniae , and hereumoniae . We propeumoniae , which ls strict . These aThe steps for epitope selection in this study mainly included selecting B-cell, MHC-II binding, and MHC-I binding epitopes. These processes were rapid, and, to improve the accuracy, more than one prediction or identification method was used. The final vaccine was a combination of conserved epitopes rather than subunit proteins, and the results of multiple sequence alignments of epitopes and their homologous sequences in different Mhp strains are shown in The AAYAAY linker was used to integrate the MHC-I/II binding epitopes to produce MhpMEV; other linkers, including AAY , GPGPG , EAAAK , and GGGp < 0.0001). IFN-γ production enhanced the activity of Th1 cells and promoted cellular immune responses, while the Th2-type immune response was mainly regulated by the secretion of IL-4 [Freund’s adjuvant is an oil adjuvant widely used in animal vaccines, but its ability to stimulate the production of antibodies against recombinant protein is not good, and it often leads to excessive inflammatory responses . Because of IL-4 ,34. CytoCurrent vaccines lack protective efficacy, and although they can reduce clinical symptoms, they cannot prevent infection by Mhp or its spreading. The strain variability may partly explain this, but local immunodeficiency due to parenteral vaccine injection also contributes ,36. In tGenome GBFF files of 21 Mhp strains were retrieved from the National Center for Biotechnology Information (NCBI) database. The files were converted to GFF3 files with BioPerl bp_genbank2gff3.pl script and used as input files for pan-genome analysis. The Roary installation and dependency checks were done on a Bio-Linux 8 system, and the pan-genome analysis was performed ,38. AfteSus scrofa (taxid: 9823) proteome using the online BLASTp program. The homologous proteins were identified as percent identity > 35, bit score > 100, and e-value < 1.0 ×10−5, and homologous proteins were removed from the core proteins [To minimize the chance of autoimmunity or an inadequate immune response, the core proteins were aligned to the proteins . For subproteins  to selecproteins  to identproteins  using thproteins . Trans-mproteins  and Signproteins , respectproteins , ANTIGENproteins , and Vaxproteins  with defproteins . ABCPred  was usedThe methods for selecting MHC-II binding epitopes were mainly based on a published study, with appropriate modifications . The B-cMHC-I binding prediction v2.23 was used to find potential MHC-I binding epitopes within the immunodominant antigens using the ANN 4.0 method . PorcineBamH I and EcoR I restriction sites of pET22b, the 6×His Tag was retained, and GenScript provided the final recombinant plasmid. E. coli BL21(DE3) competent cells were prepared by 0.1 mol/L CaCl2 treatment on ice, and the recombinant plasmid was transformed by heating at 42 °C for 90 s. The cells were plated on solid LB medium containing 20 μg/mL ampicillin for selection. For protein expression, recombinant E. coli BL21(DE3) cells were cultured in 50 mL LB medium containing 20 μg/mL ampicillin at 37 °C, and when the OD600 value reached 0.6, four culture conditions were used for MhpMEV expression: ① 0.1 mM IPTG at 18 °C for 24 h, ② 0.2 mM IPTG at 18 °C for 24 h, ③ 0.1 mM IPTG at 25 °C for 18 h, and ④ 0.2 mM IPTG at 25 °C for 18 h. After incubation, the pellets were collected and lysed by sonication on ice. The supernatant and sedimentation were separated and loaded for 12% SDS-PAGE. The immunogenicity of each amino acid in MhpMEV was calculated by BepiPred 2.0 [The MHC-I/II binding epitopes containing overlapping sequences were merged, and they were combined by the AAYAAY linker to produce an integrated recombinant protein, MhpMEV. The MHC-I binding epitopes were placed at the N-terminal and the MHC-II binding epitopes were placed at the C-terminal. For protein expression, codon optimization for MhpMEV was performed by GenScript, and the DNA sequence was synthesized. Then, it was inserted into the Pred 2.0 . The expression of MhpMEV was identified by Western blotting using a previously described method . After pE. coli BL21(DE3) was cultured in 2 L LB medium containing 20 μg/mL ampicillin. When the OD600 value reached 0.6, MhpMEV was induced by the addition of 0.2 mM IPTG and incubated at 18 °C for 24 h. The pellet was collected and lysed by sonication on ice, and the inclusion bodies were separated by centrifugation at 12,000 rpm for 30 min. Then, WB-I , WB-II (WB-I + 0.3 mol/L urea), and WB-III (WB-I + 0.6 mol/L urea) washing buffers were used to wash the inclusion bodies. The sediment was collected following centrifugation at 12,000 rpm for 10 min after each washing step. After thorough washing, MhpMEV was dissolved in 8 mol/L urea and centrifuged to remove insoluble matter. For protein refolding, MhpMEV was continuously dialyzed in a 3500 D dialysis membrane in the following dialysates: RB-I (RB-IV + 6 mol/L urea), RB-II (RB-IV + 4 mol/L urea), RB-III (RB-IV + 2 mol/L urea), and RB-IV . The time for each dialysis step was 3 h at 4 °C, and the total dialysis time was more than 12 h. After protein refolding, MhpMEV was collected following centrifugation at 12,000 rpm for 30 min. The purity and concentration were determined by BCA and 12% SDS-PAGE. MhpMEV was concentrated to 1 mg/mL by ultrafiltration and stored at −80 °C until use.Recombinant E. coli. After protein refolding, MhpMEVC6His was purified by nickel ion affinity chromatography and dialyzed in 50 mM Tris-HCl and 5% glycerol (pH 8.0) to remove imidazole. MhpMEVC6His was sent to Sangon Biotech for the circular dichroism assay. At the same time, there were no homologous sequence hits for MhpMEVC6His by the BLASTp program within the NCBI NR database, so the secondary structure was predicted by RaptorX Property [To improve the purity of MhpMEV, it was redesigned as MhpMEVC6His and expressed in Property , since iProperty  were useProperty ,61. For Property , ProSA-wProperty  and PDBsProperty  were useZDOCK  is an inAlthough the molecular docking results indicated that MhpMEVC6His could bind to immune-associated receptors, we performed animal immunization to validate our findings and studied the immune response. The design of the animal immunization was based on our previously published papers ,66,67. AThirty-two 6-to-8-week-old female BALB/c mice  were randomly divided into four groups, with eight animals per group. Grouping and vaccination strategies were as follows: ① PBS group , ② MhpMEV-25 group (each mouse immunized with 25 μg MhpMEV per immunization), ③ MhpMEV-50 group (each mouse immunized with 50 μg MhpMEV per immunization), and ④ Mhp group . For the MhpMEV-25 and MhpMEV-50 groups, the MhpMEV stock solution was diluted and emulsified with isometric Freund’s adjuvant . The emulsification was prepared according to a previously published method and used on the day of the experiment . For theFifteen 4-week-old male piglets weighing 7–8 kg were randomly divided into three groups with five piglets per group: negative control group , MhpMEV group  were mixed and emulsified, and 2 mL emulsion was administered through intramuscular injection), and Mhp group  through intramuscular neck injection on days 0 and 14). MontanideTM ISA 206 VG adjuvant consisted of water, oil, and surfactants, with high stability and only mild side effects . It is tTo detect the serum antibodies, 11 epitopes were synthesized by Sangon Biotech Co., Ltd. , with 75% purity. Recombinant proteins MhpMEV and rMhp597 and culture Mhp strain 168  were alsThe lymphocyte proliferation assay was performed on days 35 and 42 for the mouse immune experiment, as described previously . The splE. coli host as the substrate), Ca2+, and Mg2+. The rMhp597 (0.1 mg/mL) and serum samples collected on day 28 of piglet immunization were used. The reaction systems are presented in The piglet serum collected on day 28 was used to determine the antibody titer against rMhp597 according to a previously published method . To furtIn the current study, we developed a multi-epitope vaccine for MPS, named MhpMEV, based on the reverse vaccinology pipeline. The secondary structure was determined by circular dichroism, and the three-dimensional structure was predicted by Robetta, I-TASSER, and AlphaFold. The Robetta service gave a more suitable protein model, but it still showed significant differences when compared with the circular dichroism analysis results. Through molecular docking, MhpMEVC6His was able to induce immune responses. In immunized mice and piglets, MhpMEV induced humoral and cellular immune responses. Briefly, this study provides wet-lab immunoassay results of a multi-epitope vaccine derived from the reverse vaccinology pipeline and provides a conceptual multi-epitope vaccine for MPS.


Right ventricular outflow tract obstruction (RVOTO) is the most frequently encountered congenital heart disease in patients with twin –twin transfusion syndrome (TTTS) and is especially prevalent in the recipient twin. In this retrospective study, we evaluated the incidence, prognosis, postnatal management, and perinatal outcomes of and risk factors for RVOTO in the recipient twin in severe TTTS cases which diagnosed before 26 weeks after fetoscopic laser photocoagulation (FLP) at a single center in Taiwan.RVOTO was diagnosed using fetal or postnatal echocardiography. The fetal outcomes evaluated were perinatal survival rate, neonatal brain image anomalies rate, gestational age at delivery, and birth weight.p = 0.048) and had a higher percentage of cases at Quintero stage IV  than those in which the recipient twin did not have with RVOTO. Using logistic regression, we discovered that FLP at a younger gestational age  and TTTS at Quintero stage IV  were risk factors for the recipient twin developing RVOTO after FLP in severe TTTS cases.Total 187 severe TTTS cases were included; 14 (7.49%) had a recipient twin with RVOTO (12 cases of pulmonary stenosis and 2 of pulmonary atresia). Of these 14 cases, 3 (21.4%) demonstrated improvements in outflow obstruction after FLP, and 11 (78.6%) resulted in perinatal survival. Of the 11 survivors, 5 (45.5%) received transcatheter balloon valvuloplasty to alleviate the RVOTO. The perinatal survival rate, gestational age at delivery, neonatal brain image anomaly rate, and birth weights did not significantly differ between the groups in which the recipient twin had versus did not have RVOTO. Generally, the recipient twin had RVOTO received FLP at a younger gestational age . Twin–twin transfusion syndrome (TTTS) is a complication that occurs in approximately 9% of monochorionic diamniotic twin pregnancies  due to uIn TTTS survivors, the incidence of congenital heart disease can be as high as 87 per 1000 live births, a 12-fold increased incidence compared with that for singletons . Right vFetoscopic laser photocoagulation (FLP) is the first-line therapy for TTTS diagnosed prior to 26 weeks of gestation –20. BecaOur institution is the largest laser center for treatment of TTTS cases in Taiwan. In this study, we included cases of severe TTTS ( those diagnosed before 26 weeks of gestation) to analyze the incidence, prognosis, and postnatal management of and the risk factors for RVOTO in recipient twins and the influence of RVOTO in recipient twins on perinatal outcomes, including survival rate, gestational age of delivery, birth weight, and neonatal brain image results.This retrospective study was performed at the Chang Gung Memorial Hospital of Taoyuan, Taiwan, and it was approved by the Institutional Review Board of the Chang Gung Medical Foundation (202101274B0). The diagnosis of TTTS was based on ultrasound findings, as defined by Quintero et al. .A complete evaluation of the fetal structure was conducted with a Voluson Expert 8  to assess the fetal cardiac anatomy in all patients with TTTS before FLP. Color and pulsed-wave Doppler measurements were obtained in the absence of fetal breathing and movements. All cases of suspected PS or PA at the diagnosis of TTTS were referred for fetal echocardiography performed by a perinatal cardiologist. The diagnosis of PS was based on high peak systolic velocity (>90 cm/s) and turbulent flow across the pulmonary valve, with or without thickened and domed pulmonary valve cusps, which are associated with structural or functional right heart abnormalities. PA was diagnosed in the absence of forward flow across the pulmonary valve during systolic and reverse flow in the ductus arteriosus . Cases aAfter a delivery in which the recipient twin survived, the pulmonary valve pathology was confirmed in the recipient twin through postnatal echocardiography or angiographic examinations if transcatheter balloon valvuloplasty (TBV) was performed. Perinatal survival was defined as survival 28 days after delivery.Because fetal survival and intact neurological status are key outcomes for fetuses with TTTS after FLP, all living neonates with TTTS who had undergone FLP received cranial ultrasound examinations within 1 month of delivery. If two or more cranial ultrasound tests were performed, the most recent result was used. Mild cerebral image anomalies were defined as the presence of at least one of the following: intraventricular hemorrhage (IVH) of grade I or II; lenticulostriate vasculopathy; and subependymal pseudocysts. Severe cerebral image anomalies were defined as the presence of at least one of the following: IVH of grade III or IV; cystic periventricular leukomalacia of grade II or higher; porencephalic cysts; and ventricular dilatation. Ventricular dilatation was considered present when the width of the unilateral or both lateral ventricles exceeded that in the 97th percentile .t test or Mann–Whitney U test was used to compare continuous variables between groups. Qualitative data were compared using the χ2 test or Fisher’s exact test. A p value of less than 0.05 was considered significant. Logistic regression (forward condition) was used to determine the RVOTO risk factors in recipient twins. The gestational age at FLP, Quintero stage, maternal age at operation, and maximum vertical pocket of amniotic fluid (MVP) were included in to the mode when the p value for the variable was less than 0.05, and the variables were removed when the p value was greater than 0.1. The odds ratios of the variables are expressed as the odds .Statistical analysis was conducted using SPSS . The normality of the data was assessed using the Shapiro–Wilk test. The Student’s After exclusion of two cases with selective termination after laser therapy due to discordant fetal major brain image anomaly in one fetus, two cases of self-termination due to personal factors, one case of Ebstein’s anomaly in the recipient twin, one case of trisomy 21 detected after FLP, one case of donor TOF, and two cases lost to follow-up, a total of 187 cases of severe TTTS receiving FLP were included  of recipient twins after FLP. Furthermore, the incidence of RVOTO in recipient twins with perinatal survival after FLP was 7.8% (11/141), and 45.5% (5/11) of the surviving recipient twins with RVOTO required TBV to alleviate outflow obstruction symptoms. The post-FLP prognoses of severe TTTS in cases of a recipient twin with versus without RVOTO were comparable, and the two groups had similar neonatal survival rates, gestational ages at delivery, birth weights, and brain image anomaly rates.The prevalence of congenital heart disease in the general population is approximately 1% , and theThe prevalence of prenatal RVOTO occurring only after laser therapy was reported as 5.6% . As was Onset of TTTS early in gestation was reported as a risk factor for RVOTO in TTTS recipient twins, which may have been due to increased vulnerability to hemodynamic imbalances in the fetal heart in early pregnancy . We alsop = 0.78); this similarity may explain why the TTTS group with recipient twins with RVOTO had comparable outcomes, including survival and normal cranial image rates, to those of the TTTS group with recipient twins without RVOTO after FLP. Through this study, we discovered that TTTS in which the recipient twin has RVOTO was not associated with higher incidence of neonatal ultrasound brain image anomalies. In our previous study, we demonstrated that the key predisposing factor for severe brain imaging anomalies in survivors of TTTS after FLP was younger gestational age at delivery [TTTS in which the recipient twin has RVOTO after laser therapy was documented as being associated with poorer outcomes . In addidelivery . Anotherdelivery . AccordiRVOTO mainly comprises PA and PS; in our series, the incidence of PA in recipient twins with RVOTO was 14.3% (2/14). However, other studies have reported the risk of PA to be 28.5% (2/7) , 45%  of the TTTS groups in which the recipient twin had versus did not have RVOTO after FLP did not significantly differ. This may have been because of the similar percentage of high Quintero stage in the two groups and the relative low percentage of PA in TTTS cases in which the recipient twin had RVOTO.


The apparent amylose content was 21.53% in the NS. As the swelling power decreased, the percentage of soluble solids increased and different thermal properties were observed. A higher yield of the resistant starch (RS) was observed in all treated starch except PDA 25 h. The findings of our study reveal that a combination of pullulanase debranching time (15 h) and autoclaving (121 °C for 20 min) is a great technique that can be used to produce a higher amount of resistant starch in the Jinshu No.17 starch.The effects of pullulanase debranching combined with autoclaving (PDA) at various debranching times  and 121 °C/20 min of autoclave treatment on the structural and physicochemical characteristics of purple sweet potato (Jinshu No.17) starch were investigated. The results indicated that the native starch (NS) was polygonal, round, and bell-shaped with smooth surfaces. After debranching treatment, the surface of the starch samples became rough and irregular. The molecular weight became smaller after treatments. X-ray diffraction C-type pattern was transformed into a B-type structure in treated samples with increased relative crystallinity.  Ipomoea batatas (L.) Lam., is a dicotyledonous sweet potato cultivar that belongs to the Convolvulaceae family [Purple sweet potato, e family ,2. Purple family ,4. It hae family . Purple e family ,7,8, thee family ,8,9.Starch, a natural micro-crystalline biopolymer, is the most abundant reserve carbohydrate from plant organs such as seeds, fruits, leaves, tubers, and roots . Starch Currently, diverse modification approaches, such as physical, chemical, and enzymatic treatments have been reported to improve the RS content in starch. Among them, enzyme debranching of starch is the most common approach used to increase the RS3 content. Debranching starch, also called short-chain amylose or short linear glucan, is performed by treating starch paste with pullulanase or isoamylase, which causes the formation and reorganization of linear short-chain glucans under different storage conditions . PullulaTherefore, the objective of the present study was to investigate the effect of pullulanase debranching time combined with autoclaving on the structural and physicochemical properties, and in vitro digestibility of purple sweet potato starch. The structural characteristics such as morphology observed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) of starches were analyzed. The physicochemical characteristics including amylose content, swelling power and solubility, thermal property, and in vitro digestibility of starches were determined. The findings may assist in the production of new food ingredients used as RS with improved health benefits.The purple sweet potato (Jinshu No.17) variety was grown in an experimental field in Fuzhou city  and harvested in October 2020. α-amylase  and glucoamylase  were acquired from Yuanye Bio-Technology Co., Ltd. , while pullulanase (≥1000 npun/g) was obtained from Shanghai Aladdin Biotechnology . All other chemicals and reagents used in this experiment were analytical grades.The starch was extracted as described by Gou et al.,  with sliw/v) and the pH value was adjusted to 4.5–5.0 with Na2HPO4 (0.2 mol/L) buffer solution. The mixture was gelatinized in a water bath under stirring at 80 °C for 15 min. The gelatinized starch was cooled to 60 °C, and debranched by pullulanase (≥1000 npun/g) at the concentration of 10 npun/g of starch. After 0 h, 5 h, 10 h, 15 h, 20 h, and 25 h of reaction, the mixture level was adjusted to 250 mL with distilled water and the dispersion was heated in boiling water at 95 °C for 15 min to stop the pullulanase reaction. The debranched starch dispersion was then cooled to room temperature before being autoclaved for 20 min at 121 °C, re-cooled to room temperature, and stored at 4 °C for 24 h for retrogradation. The resultant was dried at 60 °C for 16 h, ground, and pass through a 100-mesh sieve. The obtained pullulanase debranching time  combined with autoclaving treatments starch was labeled PDA 0 h, PDA 5 h, PDA 10 h, PDA 15 h, PDA 20 h, and PDA 25 h, respectively.Preparation of pullulanase debranching starch was performed by a combination of the methods described by Reddy et al.  and SureA field scanning electron Microscopy system  was applied to inspect the morphology of the native and modified purple sweet potato starch. The dried starch samples were sputtered with a layer of gold prior to imaging. A micrograph of each sample was taken at an accelerating voltage of 8.00 Kv with 5000× magnification . The gra7 g/mol to 17 × 107 g/mol were used for molecular calibration.The molecular weight distributions of the native and PDA were measured using gel permeation chromatography (GPC) according to the method described by Ji et al.  with somThe crystalline structure of starch samples was analyzed using an X-ray powder diffractometer (XRD)  according to the method of Yong et al.  with som13C nuclear magnetic resonance spectrometer , using a MAS VTN 4 mm probe. The resonance frequency was 125.7 MHz, the spectral width 4 kHz, the acquisition time 50 ms and the time domain 2k. Each spectrum was scanned 2400 times. The signal intensity of the C1 region reflected the crystallized region of starch, the intensity of the C4 region reflected the amorphous region of starch, and the intensity of the C2, 3, 5 region reflected the degree of freedom of the amylose chains, as determined previously by Zheng et al. [Sample powder (0.3 g) was analyzed using a g et al. .https://connecting-asia.org/wp-content/uploads/2017/01/Rice-%E2%80%93-Determination-of-amylose-content.pdf (accessed on 30 September 2022)). The apparent amylose content was calculated as follows:A colorimetric iodine affinity procedure  multi-function microplate reader method described by Wang et al.  with somA 3 mg starch sample was mixed with 9 µL distilled water and sealed in an aluminum pan. After equilibrating for 2 h at room temperature, samples were heated from 30 to 190 °C at a rate of 10 °C/min using a differential scanning calorimetry  .−1 was used to calculate a scanning region ranging from 500 to 4000 cm−1 [A Nicolet Avatar 360 FR-IR spectrometer  was used to record the spectrum of native and modified starch samples at room temperature. Before measurement, the starch sample (0.2 g) was ground with KBr powder (150 mg) for 10 min under an infrared lamp and pressed into tablets. The average of 32 scans at a resolution of 4 cm000 cm−1 .Starch sample (0.5 g) was mixed with 25 mL of distilled water and heated at 65, 75, 85, and 95 °C in a water bath for 20 min and stirred every 5 min. The samples were cooled to room temperature, and the suspension was centrifuged at 3500 rpm for 20 min. The supernatant and sediment samples were separated and weighed, and the swollen starch sediment was weighed after an aliquot of the supernatant was evaporated at 105 °C for 2 h . The equThe Jinchu No.17 starch fractions in the samples were determined by previously described method of Thanh et al.  with somp < 0.05, significant difference). All experiments were repeated at least in triplicate measurements.Graphs were constructed in OriginPro 2021 . IBM SPSS  was used for the experimental data analysis, and one-way ANOVA was performed to test for significant differences (The morphology of native and debranching combined with autoclaving starches prepared from Jinshu No.17 is shown in 6 g/mol) was observed in the NS. After PDA treatment, the samples showed a moderate and broad molecular distribution with lower Mn and Mw after modification under different treatment times compared to NS. This indicates that purple sweet potato starch has been broken down into low-polymerization debranching starch. The polydispersity values (PD) of all treated samples increased, indicating a larger molecular weight dispersion following debranching times. This indicates that the purple sweet potato starch was digested by pullulanase to prepare starch with a smaller molecular weight. The degraded products of the starch samples entered the gel and were eluded together to form an amylose peak, thereby increasing the percentage of resistant starch [Gel permeation chromatography (GPC) technique was used for analyzing the polymer of starch samples to determine their molecular weight (Mw), number average (Mn), and polydispersity values . The molt starch . After 2a-type crystalline polymorph [b-type and A-type. After samples were subjected to pullulanase debranching time combined with autoclaved treatment and retrograded, all the X-ray diffraction patterns of starches was transformed to a B-type structure with a principal sharp peak (2θ) at 17° and some small additional peaks at around (2θ) values of 14.96, 22.12, and 23.92° which were characterized as B-type crystallinity [olymorph ,8. Howevolymorph  reportedolymorph  reportedallinity . The chaallinity ,42. As sallinity .The nuclear magnetic resonance spectra of pullulanase debranching with autoclave treatment on purple sweet potato starch were assigned as described previously and are displayed in The apparent amylose content (AC) of native purple sweet potato starch was found to be 21.53% . The amyThe DSC of the NS and PDA of Jinshu No.17 starch samples is shown in −1 for stretching vibration of O-H groups, at 2922 cm−1 for stretching vibrations of C-H and CH2, at 1645 cm−1 for H-O-H bending of water molecules adhered to NS and PDA at different times, at 1156 cm−1 for asymmetric stretching vibration of C-O-C, at 1026 cm−1 for symmetric stretching vibration of C-O-C. The characteristic peaks of both NS and PDA at approximately 3035–3710 cm−1 shifted to a higher frequency, indicating that pullulanase debranching treatment times with greater R spectra values promote higher levels of order in the external regions of the starch granule [The FT-IR spectrum can reflect the short-range ordered structure in the starch external region through vibrational deviations upon interactions between native and modified starches.  granule ,43.Dioscorea alata L [The swelling power and percentage of soluble solids of NS and PDA starches are presented in  alata L . It has  alata L . TherefoThe RDS, SDS, and RS content in the Jinshu No.17 NS and PDA starch samples are presented in The combination of pullulanase debranching time and autoclaving (PDA) has a significant effect on the structural, physicochemical characteristics, and in vitro digestibility of purple sweet potato starch. Compared with NS, the morphology of starch structure was destroyed with significantly different particle sizes after all debranching treatment times. Different decreases in the molecular weight were observed and the XRD revealed a change from C-type to B-type crystalline pattern when debranched and autoclaved, thus improving the regularity of the crystallites by enhancing RS yield. Analysis using NMR indicated an increase of the double helix and higher degree of freedom of amylose chains. The apparent amylose content and the thermal property of starch were greatly influenced by the duration of PDA. The FT-IR showed that there was no more change between the NS and PDA samples. The swelling power increased with the temperature and decreased with the debranching times, while the percentage of soluble solids increased. Higher RS content (70.14%) was obtained after 15 h of pullulanase debranching combined with autoclaving (121 °C/20 min). In comparison to the starch sample at 15 h, the damage after 20 h decreased RDS, increased SDS, while RS was low. The results demonstrated an increase in the RS, but it was not stable across treatment times. Pullulanase debranching times combined with autoclaving is a promising green and clean technique for the preparation of RS3. Therefore, purple sweet potato starch produced with PDA could be used as an ingredient in foods and industrial products to provide biological or physical functionalities.


A novel type of phenol-formaldehyde (PF) resin was prepared by utilizing the liquefaction products liquefied by phenol under acidic conditions and then reacted with formaldehyde under alkaline conditions. The relationship between the liquefaction behavior of cassava starch and the properties of modified PF resin wood adhesive was studied. The effects of the liquid–solid ratio of phenol to cassava starch, sulfuric acid usage, and liquefaction time on the liquefaction residue rate and relative crystallinity of cassava starch were determined. The results showed that the bonding strength of modified PF resin decreased gradually with the decrease of the liquid–solid ratio. It was a great surprise that bonding strength still met the requirement of the national standard of 0.7 MPa when the liquid–solid ratio was 1.0. The detailed contents were analyzed through FT-IR, SEM, and XRD. In terms of the utilization of bio-materials for liquefaction to synthesize wood adhesive, cassava starch may be superior to the others. The utilization of biomass resources to produce biofuel or other chemical products is no longer unusual now ,3,4,5,6.In the wood adhesive industry, starch has been applied to prepare starch-based adhesives for a long time ,14,15. ABiomass liquefaction is a kind of technology in which biomass materials are utilized efficiently and comprehensively. A lot of biomass materials have been applied to the synthesis of wood adhesives, such as wood , lignin At present, relevant reports about the utilization of starch, especially cassava starch, to be liquefied for the sake of modifying PF resins are very few. The principal purpose of this study is to discuss the effects of liquefaction conditions on the liquefaction products of cassava starch liquefied by phenol under acid conditions and its residue rate. The relationship between cassava starch liquefaction behavior and the properties of cassava starch-based phenol-formaldehyde resin (CPF). This work is also expected to provide support and reference for the investigation and optimization of starch or other carbohydrates in the future.Phenol  and formaldehyde solution  is used for preparing PF and CPF resins, were purchased from Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China. Cassava starch, the main raw material for preparing CPF, was brought from Zhenlong Starch Co., Ltd., Chongzuo, China. Sodium hydroxide solution (40% of concentration) is used for adjusting the pH value during the process of preparing PF and CPF resins was also brought from Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China. Besides, sulfuric acid  as a liquefied catalyst for cassava starch was obtained from Chengdu Cologne Chemical Reagent Factory, Chengdu, China. Eucalyptus veneers  are used for preparing plywood that was purchased from Zhenshuo Wood Industry Co., Ltd., Nanning, China.Liquefaction parameters were fixed as the mass ratio of phenol to cassava starch was 2:1, liquefaction time was 2 h, liquefaction temperature was 120 °C, and acid catalyst usage was 8% of the mass of cassava starch. At first, measured phenol (250 g) and acid catalyst (12.5 g) were added into a four-necked flask in order. At this time, the reflux condenser and motor-driven stirrer were turned on. Secondly, pre-weighed starch (125 g) was poured into the flask when the liquefaction temperature reached 120 °C. Lastly, the temperature of the reaction system was quickly cooled down to 70 °C, and an appropriate amount of NaOH solution  was slowly added to the flask to adjust the pH of this system to 7.0 and kept the reaction temperature for 60 min, the black and homogeneous liquefied products of cassava starch were obtained eventually.M0 is the weight of residue after liquefaction (g); M1 is the weight of lignin before liquefaction (g).Quantitative liquefaction products of cassava starch were diluted and dissolved with acetone, which was pumped through filtration by a Buchner funnel. Continue to flush the funnel with acetone until it becomes colorless. The residue was put into a thermostatic drying oven and dried at 105 °C to constant weight. The residue rate was calculated according to Equation (1).The first batch of weighed formaldehyde solution (252 g) was added to the liquefied products prepared by 2.2.1. Then NaOH solution  was added and the reaction temperature slowly heated to 90 ± 2 °C within 50–60 min. The temperature was lowered to 60 °C after 15 min, then the remaining formaldehyde solution (223 g) was added to the system, and the temperature was slowly raised to 90 ± 2 °C within 50–60 min. Eventually, the black and homogeneous CPF resin can be obtained when the viscosity reaches 65–100 mPa·s (25 °C) under these conditions.Phenol (252 g) was added into a four-neck flask and the blender was turned on at this time. Then PF resin was synthesized by repeating the steps described in 2 to press three layers of plywood. Hot-pressing parameters: prepressing time was 30 min, hot-pressing temperature was 150 °C, hot-pressing pressure was 1.2 MPa, and hot-pressing time was 65 s/mm.Eucalyptus veneers with a moisture content of 5–8%, thickness of 1.7 mm, and width of 480 × 480 mm were selected. The prepared wood adhesive (64 g resin mixed with 9.6 g flour) was applied to veneers with the consumption of 332 g/mBonding strength tests were carried out on microcomputer-controlled electronic universal test machines according to the method described in “GB/T 17657-2013, Test methods of evaluating the properties of wood-based panels and surface decorated wood-based panels”. The prepared plywood was cut into sizes of 100 × 25 × 3.25 mm at first, and then these cut test pieces were put into boiling water for about 3 h and then air-dried for 20 min. In order to guarantee the validity of test results, the number of specimens to be tested was at least ten for every piece of plywood and took the average value.−1–450 cm−1, resolution was 4 cm−1, and scanning times was 64.The resins (8–10 mg) and liquefied products (8–10 mg) were freeze-dried at −70 °C for 8 h and then vacuum dried for 16 h and placed into a Nicolet I S 50 Fourier transform spectrometer after tabletting. Measuring parameters: air as scanning background, the crystal was a highly sensitive diamond, scanning range 4000 cmThe liquefied product powders (0.1–0.2 g) were ground in a mortar to a size less than 100 meshes and put on quartz glass. The liquefied product powders on the surface of quartz glass were flattened evenly with a glass plate and the residual powders outside the quartz glass were wiped with absorbent paper. Afterward, the quartz glass was fixed on the sample stage of the console. The scan range was from 5 to 50 deg, the scan rate was 4 deg/min, and the step size was 0.02 deg.Conductive tape was stuck on the sample table at first, and then the PF or CPF resin or liquefied product powders (0.1–0.2 g) were evenly sprinkled on the conductive tape. The powders that were not stuck were blown with compressed air  to prevent the contamination of the equipment by the powders that were not stuck, and the powders were gold-coated to strengthen conductivity. Afterward, prepared specimens were put on the sample stage. The buttons of of the pump and beam were turned on in order and the test started.Generally speaking, the residue rate of biomass materials after liquefaction depends on the liquefaction temperature, liquefaction time, liquid–solid ratio, and catalyst to a great extent. A strange phenomenon was found that the interior temperature of the reaction system was hard to continue increasing when it reached 115 °C in the case of cassava starch. The interior temperature of the reaction system was no longer increased even though the exterior temperature increased to 180 °C. It may be a combination of disadvantages of the conventional heating process and cassava starch itself. Therefore, liquefaction temperature was fixed at 120 °C and the effect of liquefaction temperature on residue rate did not discuss in this section. + attacking the components of cassava starch was increased, and the efficiency of mass transfer and heat transfer was improved, which made the reaction carried out under the conditions as homogeneous as possible. On the other hand, phenol is a strong polar solvent, and the higher the liquid–solid ratio, the better the dispersion and swelling of cassava starch, thus, the solubility of liquefied products was increased and the residue rate was reduced [As clearly shown in  reduced . In addi+ has a great influence on the liquefaction reaction [+ in the liquefaction system is sulfuric acid, followed by phosphoric acid, and oxalic acid is the lowest according to their dissociation constants [As the catalyst in the liquefaction of biomass materials, the most used catalysts were mineral acids such as sulfuric acid, hydrochloric acid, and other strong acids or phosphoric acid, oxalic acid, and other weak acids. In general, the liquefaction reaction progresses smoothly in the presence of strong acids while it is slow and incomplete in the presence of weak acids . It can reaction . A conclonstants . Therefoonstants . ConsequWhen it comes to the effect of liquefaction time on residue rate, the recondensation reaction of liquefaction products ought to be taken seriously. As is known to all, the liquefaction reaction consists of the degradation and recondensation of biomass materials. The molecules of liquefaction products of cassava starch would be polymerized and then increase residue rate if the liquefaction time was further prolonged under the same conditions, which is a spontaneous chemical reaction . Figure The amorphous region of starch granules is comprised of amylose associated with large branches of amylopectin molecules, and the crystalline region is formed by amylopectin molecules with short branches . The liq+ was beneficial for destroying the crystal morphology of cassava starch and decreasing the residue rate of that once again. Besides, the appropriate liquefaction time was good for the destruction of crystal morphology of cassava starch, whereas the excessive liquefaction time may cause the recondensation of liquefaction products, which directly increased crystallinity. It also proved that excessive liquefaction time has a passive influence on the improvement of the liquefaction of cassava starch. And the relative crystallinity was decreased with the increase in liquid–solid ratio, which indicated that the increase in phenol can make cassava starch fully liquefy. The liquefaction of cassava starch is often divided into two phases under the cooperation of acid and phenol: liquefaction preferentially attacks the amorphous region of the particles with a high reaction rate in the early stage, and the particles of the crystalline region are liquefied at a slower rate in the subsequent stage. The decline of the relative crystallinity of cassava starch is a benefit for improving its activity and offering a foundation for the reaction with other substances.In order to better understand the effect of a single liquefaction parameter  on the crystallization morphology of the liquefaction products, a single factor experiment was carried out, and the results are shown in −1, which was the characteristic absorption of the bending vibration of the C-H bond of glucose. It was suggested that the molecular structure of cassava starch was damaged to some extent, and the smaller molecular fragments of cassava starch had an electrophilic addition reaction with the benzene ring, which was the key to the formation of different substituted compounds of the benzene ring [−1, the peak of the liquefaction products was stronger than that of cassava starch. The vibration of an aromatic skeleton at 1515 cm−1 and stretching vibration of C=O at 1602 cm−1 were observed [−1 had been widened, which may result from the reaction of cassava starch and phenol under acidic conditions. The molecular weight was decreased and the number of unsaturated bonds was increased, which changed the structure of cassava starch and made a large number of hydroxyl groups exist in the liquefied products.As shown in ene ring . In the observed . Therefo−1 and 1696 cm−1, which stated the presence of methyl and conjugated carbonyl groups existed in the liquefaction products. The peaks at 1602 cm−1 and 1225 cm−1 became sharper with the increase in catalyst usage, which meant that the chemical compositions of cassava starch changed more dramatically when the acid conditions were strengthened, and more aromatic nuclear derivatives with reactive activity were produced. −1 was increased slowly with the increase in liquefaction time, but the trend was not obvious. The intensity of wave peaks at 1602 cm−1 and 1225 cm−1 was enhanced with the increase in liquefaction time, which was similar to the influence of different catalyst usage. −1, 1696 cm−1, 1602 cm−1, and 827–1225 cm−1. They all became sharper with the increase in the liquid–solid ratio, which suggested the increase in phenol is beneficial for improving the activity of cassava starch.As can be seen from −1 was strengthened. It may be the characteristic absorption of out-of-plane bending vibration of aromatic C-H bond after the 1,2 disubstitution of the benzene ring. And there were stretching vibrations of the C-H bond after 1,2,4 substitutions of the benzene ring at 824 cm−1, which indicated that compounds with different substituents might be formed on the aromatic ring. Therefore, it could be inferred that substitution reactions occurred on the benzene ring. The peak pattern was different at 1014 cm−1, where was the stretching vibration of C-O of aromatic ether, and at 1147 cm−1 was the stretching vibration of C-O-C of aromatic ether [−1 [−1 was C=O stretching vibration and aromatic skeleton vibration [−1, and O-H stretching vibration was observed at 3253 cm−1. It may result from phenol reacting with cassava starch to generate liquefied products, thus resulting in the decrease of phenol that is directly involved in the formaldehyde reaction.As is vividly demonstrated in ic ether . It can ther [−1 . Compareibration . The liqThe SEM pictures of cassava starch, liquefied products, and PF and CPF resins are clearly shown in The liquefaction products of cassava starch, a kind of alternative to phenol, presented a certain potential in the modification of PF resins. Significantly, the wet bonding strength of CPF resin was hardly changed when the liquid–solid ratio exceeded 2.0:1. The further increase in the liquid–solid ratio had made less sense for the improvement of the water resistance of CPF resin. The liquefaction reaction of cassava starch tended to complete when the liquid–solid ratio was 2.0:1. And the overall activity of the system was higher at this time, thus, the quality of CPF resin prepared by the reaction with formaldehyde was also higher. Of course, the increase in the relative content of phenol was another reason for that. Admittedly, the adhesive properties of CPF resin still have a certain distance to PF resin, but not much. It can be seen that cassava starch is inherently poor water resistance and is capable of preparing wood adhesive that meets the requirement of national standards. In addition, as for the production cost and environmental protection, CPF resin is superior to PF resin. Therefore, choosing cassava starch as the raw material to produce CPF resin has a positive significance for prolonging the related industrial chains and improving social and economic benefits.In the current work, the cassava starch was liquefied by phenol under acidic conditions, then reacted with formaldehyde under alkaline conditions, the novel type of PF resin was synthesized successfully. Compared with PF resin, the introduction of liquefaction products of cassava starch provides CPF resin with almost bonding strength and lower formaldehyde emission under the same reaction conditions. The utilization of renewable resource cassava starch to partially replace phenol and formaldehyde plays the role of alleviating resource pressure and mitigating environmental pollution. However, optimal liquefaction parameters for cassava starch have yet to be exploited. More single-factor experiments are needed, and a mature orthogonal experimental design should be formulated. More instruments such as NMR, XPS, GPC, and TEM ought to be utilized for the exploration of the detailed liquefaction mechanism of cassava starch. In addition, the thermal properties of CPF resin should be studied systematically.


Objective: To investigate the flexural strength of hot isostatically pressed nano 3 mol% yttria partially stabilised zirconia and conventionally sintered micro 3 mole% yttria partially stabilised zirconia. Methods: A total of 40 bar-shaped (2 mm × 4 mm × 16 mm) specimens were prepared from hot isostatically pressed nano 3 mol% yttria partially stabilised zirconia  and conventionally sintered micro 3 mole% yttria partially stabilised zirconia . Two groups were prepared for each material (n = 10), with one serving as ‘control’ and the other being cyclically loaded using a chewing simulator : SEM imaging was also undertaken on selected specimens. Flexural strength until fracture was recorded (ISO 6872). Paired and unpaired t-tests were chosen to compare mean outcomes between the four groups (p < 0.05). Results: Significant statistical difference was only found between the means of control specimens. CeramaZirc Nano HIP had the highest mean value (1048.9 MPa), whilst the lowest was seen for CeramaZirc after loading (770 MPa). Values for both materials were higher without loading than after loading. Values after cyclical loading showed large SD values (276.2–331.8) in comparison to ‘control’ (66.5–100.3). SEM imaging after cyclical loading revealed a smoother and less destructed surface of CeramaZirc Nano HIP compared to CeramaZirc. Significance: HIP nano zirconia exhibited inferior strength, surface polishability and behaviour to loading. Therefore, further investigation on the behaviour of such materials should be conducted before recommending for clinical use. Zirconium dioxide (ZrO₂), known as zirconia, is a white crystalline oxide of zirconium’. Nevertheless, pure zirconium does not exist in nature, ‘but only in conjunction with silicate oxides (ZrO₂ × SiO₂) or as free oxide (ZrO₂)’ . MoreoveZirconium was first introduced in dentistry as an endodontic post and implant abutment in the 1990s, and its use expanded afterwards . That isThe use of zirconia and zirconia-reinforced restorations has increased over the last decade, especially with a drive for more aesthetically pleasing restorations . HoweverZirconium has many crystalline forms: monoclinic, tetragonal, hexagonal and cubic polycrystals. Cubic and tetragonal polycrystals are able to be stabilised at room temperature with the addition of metal oxides during firing; the most used stabiliser is yttrium . AdditioContinuous development of zirconium materials for dental use may revolve around nano-grain size with superior mechanical properties and upgraded translucency : it has CeramaZirc Nano HIP is a recently introduced non-dental pressed nano zirconia, which combines nano and pressing technology.The aim of this study was to evaluate the strength pre- and post-loading of a recently introduced polycrystalline zirconia by comparing the flexural strength before and after chewing simulation between nano HIP 3 mol% yttria partially stabilised zirconia and conventionally sintered micro 3 mole% yttria partially stabilised zirconia to investigate the effect of hot pressing and nanotechnology on the behaviour of polycrystalline zirconia if pressable zirconia ingots to be used in dentistry. The Null hypotheses were: (i) there is no statistically significant difference between the flexural strength of conventional zirconia and HIP nano zirconia; (ii) there is no statistically significant difference between the pre- and post-loading flexural strength of conventional zirconia and HIP nano zirconia.Two materials were selected: hot isostatically pressed nano 3 mol% yttria partially stabilised zirconia  and conventionally sintered micro 3 mole% yttria partially stabilised zirconia . Nano HIP has been recently developed, and at the time of the study was not yet commercially available. The mechanical properties according to the manufacturer are listed in Both materials were fabricated and prepared by the manufacturer. A spray-dried powder was pressed to net shape in a die mould. This compact was then cold isostatically pressed at room temperature in a wet isostatic press (WIP) at high pressure, followed by sintering at high temperature (1550 °C) where the compact shrinks at approximately 20% and becomes ultra-hard; CeramaZirc was then cut from this. The rest of the tile (once cool), was then hot isostatically pressed (HIP) at 1450 °C to make CeramaZirc Nano HIP. Finally, both materials were diamond ground (126 µ grit) to form 40 bars (20 of each) of 2 mm thickness, 4 mm width and 16 mm length (ISO 6872). Specimens for each were divided into 2 groups: CeramaZirc Nano HIP ; CeramaZirc . Specimens were dry stored before mechanical testing.A 3-point flexural strength test was conducted on M1 and N1 groups, using a Universal Testing Machine  and TesXpert V11.02 software.P is the breaking load, in N;l is the test span, in mm;w is the width of the specimen, in mm;b is the thickness of the specimen, in mm.The machine was set as follows: 2 kN loading cell; 1000 mm softened upper switch; 200 mm lower softened switch; 1700 N upper force limit; 100 N lower force limit; 10 N pre-load; 1 mm/min loading arm speed (ISO 6872); 12 mm holding arms distance. The following equation was used to calculate flexural strength values:Cyclic loading was undertaken for M2 and N2 groups using a chewing simulator CS-4.2  . The macThe specimen cup was filled with silicone  to form a mould and to simulate the cushioning effect of the periodontal ligament. The mould was formed by filling the cup with a silicon mix. Before the silicon was set, a rectangular acrylic piece (25 mm × 13 mm × 3 mm) was seated into the silicone with its surface flush with the silicon surface. Once the silicon was set, the acrylic piece then removed leaving its negative imprint, which was filled later with a clear orthodontic acrylic powder and liquid , which once it was set, it formed a slab.Afterwards, the acrylic slab was heat-cured under 2 bar pressure at 45 °C for 8 min . The clear acrylic slab was finished and polished using an acrylic grinder  and a rubber polisher  underwater, then sandblasted along with the specimens in groups M2 and N2 with 110µm Al₂O₃ particles  at a pressure of 2 bar , at a distance of ~10 mm, at 90°, for 10 s using a linear motion. A separate acrylic slab was made for each test specimen.The zirconia specimens for each group were positioned in the middle and adhesively cemented onto the acrylic slabs using Panavia F 2.0  following the manufacturer instructions for cementing zirconia restorations, and light-cured for 20 s from each side . Once the specimens were adhesively cemented onto the acrylic slabs, slabs were seated back to their negative imprints in the mould cups. The chewing simulator machine has two chambers; each chamber can house a single cup as shown in After chewing simulation, to prepare the specimens for SEM scanning and post-cyclic loading mechanical testing, they were detached from the acrylic slabs using an 11 mm hollow acrylic grinder  to remove the acrylic slab around the specimen and a grey torpedo rubber pre-polisher to remove the remaining thin cement layer .3 density for 120 s at room temperature , followed by silver painting around the specimens to reduce charging. The specimens were cleaned then with 50 psi air pressure. The specimens were scanned with SEM using secondary electrons (SE) at 5 eV  at different magnification .After cyclic loading, one sample of M2 and N2 were placed on carbon tabs and mounted on aluminium stubs . 10 nm At-tests were chosen to compare mean outcomes between the groups (p < 0.05). Normality and equal variance were verified (Levene’s test > 0.05). Box and whisker plots used to compare the range of values spread between groups and to spot outliers , but not between M2-N2 groups . The second null hypotheses was accepted as there was no significant difference between paired groups for pre- and post-loading flexural strength; however the post-load behaviour was notably different with a much broader range of failure.The first null hypothesis was rejected, as conventional zirconia exhibited a high and consistent statistically significant pre-loading strength compared to HIP nano zirconia  as illustrated in HIP process (‘post-HIP’ or ‘post-sinter HIP’) has been claimed to improve mechanical properties due to overcoming/reducing manufacturing-related defects . In the It has been shown that polished zirconia restorations lead to less wear of antagonistic teeth than feldspathic porcelain, lithium disilicate and nickel-chromium restorations ,34,35. WAlthough HIP nano zirconia exhibited lower strength than conventional 3 mole% zirconia, its strength is equivalent to conventional 4 mole% zirconia, which is suggested as an option for the fabrication of up to 3-unit posterior fixed bridges ,41,42,43Conventional micro zirconia exhibited high flexural strength.Conventional micro zirconia has shown less particle debris around the impact point after loading, indicating higher structural integrity and a smoother surface, suggesting higher polishability.Nano HIP zirconia and conventional micro zirconia were affected significantly after cyclic loading, and are not recommended for use in bruxists.Within the limitations of this study, the following was concluded:


Filobasidium genus were assembled and compared with other Tremellomycetes mitogenomes. The mitogenomes of F. wieringae and F. globisporum both comprised circular DNA molecules, with sizes of 27,861 bp and 71,783 bp, respectively. Comparative mitogenomic analysis revealed that the genetic contents, tRNAs, and codon usages of the two Filobasidium species differed greatly. The sizes of the two Filobasidium mitogenomes varied greatly with the introns being the main factor contributing to mitogenome expansion in F. globisporum. Positive selection was observed in several protein-coding genes (PCGs) in the Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina species, including cob, cox2, nad2, and rps3 genes. Frequent intron loss/gain events were detected to have occurred during the evolution of the Tremellomycetes mitogenomes, and the mitogenomes of 17 species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina have undergone large-scale gene rearrangements. Phylogenetic analyses based on Bayesian inference and the maximum likelihood methods using a combined mitochondrial gene set generated identical and well-supported phylogenetic trees, wherein Filobasidium species had close relationships with Trichosporonales species. This study, which is the first report on mitogenomes from the order Filobasidiales, provides a basis for understanding the genomics, evolution, and taxonomy of this important fungal group.In the present study, two mitogenomes from the The online version contains supplementary material available at 10.1186/s43008-022-00094-2. Indeed, although there are about 1 million species of basidiomycetes, there are less than 120 basidiomycetes mitogenomes available in the database. Basidiomycetes are widely distributed worldwide and play important roles in industry, medicine, agriculture, food supply, and maintenance of forest ecosystems  reveal the genetic characteristics of Filobasidium mitogenomes; (2) characterize the evolutionary dynamics of introns in Tremellomycetes mitogenomes; and (3) determine the phylogenetic placement of Filobasidium in the phylum Basidiomycota based on the combined mitochondrial gene dataset (15 core PCGs). This is the first report of Filobasidium mitogenomes. The availability of these mitogenomes will improve our understanding of the genetics, evolution, and taxonomy of Filobasidium species and other closely related fungal species.In the present study, the mitogenomes of two F. wieringae and F. globisporum mitogenomes assembly were downloaded from the Sequence Read Archive database . Quality control steps conducted to remove unqualified sequences from the raw sequencing data consisted of filtering low-quality sequences using ngsShoRT 2.2 . Strand asymmetries of Tremellomycetes mitogenomes were calculated based on the following formulas: AT skew = [A − T]/[A + T], and GC skew = [G − C]/[G + C]. Codon usages of the two Filobasidium mitogenomes were analyzed using Sequence Manipulation Suite (Stothard Ka) and synonymous (Ks) substitution rates for core PCGs in the 17 mitogenomes from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina were calculated using DnaSP v6.10.01  in the 17 mitogenomes were analyzed using MEGA v6.06 based on the Kimura-2-parameter (K2P) substitution model  of the two Filobasidium mitogenomes against themselves  according to the method described by Férandon et al.  in the corresponding reference gene. When the same Pcls are present, they are considered orthologous introns and usually have high sequence similarity  in a 50% majority-rule consensus tree  to investigate the phylogenetic status of F. wieringae and F. globisporum were deposited in the GenBank database . In addition, the mitogenome of F. globisporum contained four non-conserved PCGs encoding proteins with unknown functions and three non-conserved PCGs encoding GIY endonucleases. The F. globisporum mitogenome contained 37 introns distributed in the atp9, cob, cox1, cox2, cox3, nad1, nad4, nad5, rns, and rnl genes, of which 27 belonged to group I, two belonged to group II, and eight were of unknown types. A total of 26 intronic ORFs were detected in these introns, encoding LAGLIDADG homing endonucleases, GIY-YIG homing endonucleases, and proteins with unknown functions. Seven introns were detected in the mitogenome of F. wieringae, six of which belonged to group I. No intronic ORFs were detected in introns of F. wieringae.The structures of the two Filobasidium mitogenomes contained two rRNA genes, namely the small subunit ribosomal RNA (rns) and the large subunit ribosomal RNA (rnl) , followed by the D arm.Both F. wieringae across the neighboring genes nad2 and nad3 (− 1 bp), as well as between nad4L and nad5 (− 1 bp) . A total of 6870 bp and 10,849 bp of intergenic sequences were detected in the mitogenomes of F. wieringae and F. globisporum, respectively. The length of these intergenic sequences ranged from 16 to 1291 bp. The longest intergenic sequence was located between nad5 and trns in the F. globisporum mitogenome.Two overlapping nucleotides were detected in the mitogenome of d nad3 −  bp, as wF. wieringae mitogenome (48.40%) . The F. wieringae mitogenome was 43,922 bp smaller than that of F. globisporum. Intronic regions made the greatest contribution to the size expansion of the F. globisporum mitogenome, with a contributing rate of 81.48%. Protein coding regions accounted for 9.56% of the size variation, while intergenic regions contributed 9.06% of the F. globisporum mitogenome expansion.The protein coding regions accounted for the largest proportion of the 0%) Fig. , while tAgaricomycotina, Pucciniomycotina, and Ustilaginomycotina mitogenomes used ATG as the start codon, except for the cox1 gene of Ustilago bromivora and the cox1 and nad2 genes of Ustilago maydis, which used GTG, and the cox2 and rps3 genes of Jaminaea angkorensis, which used TTG , GAA , TGT , AAA , GAT , and CAT  .Codon usage analysis indicated that the most frequently used codons in the two is) Fig. . The freFilobasidium mitogenomes against themselves and identified 0 and 16 repeat sequences in the mitogenomes of F. wieringae and F. globisporum, respectively  genetic distance between the 17 species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, followed by the nad3 and nad6 genes, demonstrating that these genes had differentiated greatly during evolution  among the 15 detected core PCGs, while nad4L had the smallest Ka value. The synonymous substitution rate (Ks) of the nad1 gene was largest, while that of the cox2 gene was smallest among the 17 species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina. The Ka/Ks values for most of the 15 core PCGs were < 1, indicating that these genes were subjected to purifying selection pressure. However, the average Ka/Ks values of the cob, cox2, nad2, and rps3 genes were > 1, indicating these genes might have been subjected to positive selection.Among the 15 detected core PCGs, the ion Fig. . The nadrns, rnl, atp6, atp9, cob, cox1, cox2, cox3, nad1, nad3, and nad5 genes of the 17 mitogenomes from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina  using the cox1 gene of the medical fungus Ganoderma calidophilum  were only detected in one of the 17 species. However, some rare introns in Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, including P237, P900, P1030, and P1057, were detected in distantly related species, such as Hygrophorus russula  was the fourth largest among the 17 mitogenomes from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, which was only smaller than that of Ustilagobromivora  from the order Ustilaginales, Microbotryum cf. violaceum , and Microbotryum lychnidisdioicae  from the order Microbotryales. The GC content of the 17 mitogenomes ranged from 28.79 to 40.43%, with an average of 34.15%. The GC content of the two Filobasidium species was much higher than the average value. Eight and five of the 17 mitogenomes from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina had positive AT skews and GC skews, respectively. Each mitogenome contained 15–51 PCGs, and the mitogenome of Microbotryum lychnidisdioicae contained the most PCGs. The mitogenome of F. globisporum contained the greatest number of introns (37) and intronic ORFs (26) among the 17 mitogenomes detected. All 17 mitogenomes contained two rRNA genes. In addition, 20–31 tRNA genes were detected in the17 species from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina.The sizes of 17 mitogenomes tested varied greatly, ranging from 24,874 to 177,540 bp, with an average size of 46,800 bp   Fig. . All majFilobasidium species varied in length and base composition. In addition, possible positive selections on cob, cox2, nad2, and rps3 genes were detected between some species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina. Core PCGs, including atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6, are used for energy metabolism, and the rps3 gene is likely involved in assembly of the mitochondrial small (37S) ribosomal subunit , indicating that the mitochondrial gene is an effective tool for analysis of the phylogenetic relationship of basidiomycetes. More mitogenomes are needed to promote the classification or identification of basidiomycetes and reconstruct the phylogeny of fungi.Basidiomycetes are a diverse group that are distributed worldwide. Basidiomycetes play an important role in industry, medicine, agriculture, and ecological maintenance (Alves et al. Filobasidiales order were reported and compared with other mitogenomes from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, including F. wieringae and F. globisporum. The mitochondrial genome size of the two Filobasidium species varied greatly, ranging from 27,861 to 71,783 bp, and the intronic region was considered to be the main factor contributing to mitogenome size variations in the Filobasidium genus. We further found intron loss/gain events in Tremellomycetes and other mitogenomes occurred during evolution. Comparative mitogenomic analysis revealed that the genetic contents, codon usages, and repetitive sequence differentiated greatly in the two Filobasidium species. In addition, a large number of base and sequence length variations were found in the core coding genes, tRNA genes and rRNA genes of the two Filobasidium mitogenomes. Several core PCGs have experienced strong pressure of positive selection in mitogenomes from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, including cob, cox2, nad2, and rps3 genes. In addition, large-scale gene rearrangements were detected between the 17 species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina, which showed that the mitochondrial gene arrangement was highly variable. We further analyzed the phylogenetic status of Filobasidium species based on BI and ML methods using a combined mitochondrial gene set. This study serves as the first investigation of mitogenomes from the order Filobasidiales, and the results presented herein will help improve our understanding of Filobasidiales genomics, evolution, and taxonomy.In the present study, two complete mitogenomes from the Additional file 1: Table S1. Comparison on mitogenomes among 17 species from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. Table S2. Annotation and characterization of the two Filobasidium mitogenomes. Table S3. Core protein coding gene information of the two Filobasidium species. Table S4. Start and stop codons analysis of 17 species from Agaricomycotina, Pucciniomycotina and Ustilaginomycotina. Table S5. Local BLAST analysis of the Filobasidium mitogenomes against themselves. Table S6. Tandem repeats detected in the mitogenomes of Filobasidium using the online program Tandem Repeats Finder. Table S7. Species, and GenBank accession number used for phylogenetic analysis in this study.Additional file 2: Figure S1. Putative secondary structures of tRNA genes identified in the mitochondrial genomes of two Filobasidium species. The 22 tRNAs in green or red fonts represent tRNAs shared by the two Filobasidium species, while the tRNA in blue font represent tRNA only in F. wieringae. Residues conserved across the two mitochondrial genomes are shown in green, while variable sites are shown in red. All genes are shown in order of occurrence in the mitochondrial genome of F. wieringae, starting from trnL.