Frizzled (FZD) proteins function as receptors for WNT ligands. Members in FZD family including FZD2, FZD4, FZD7, FZD8 and FZD10 have been demonstrated to mediate cancer cell epithelial-mesenchymal transition (EMT).CCLE and TCGA databases were interrogated to reveal the association of FZD5 with EMT. EMT was analyzed by investigating the alterations in CDH1 (E-cadherin), VIM (Vimentin) and ZEB1 expression, cell migration and cell morphology. Transcriptional modulation was determined by ChIP in combination with Real-time PCR. Survival was analyzed by Kaplan–Meier method.In contrast to other FZDs, FZD5 was identified to prevent EMT in gastric cancer. FZD5 maintains epithelial-like phenotype and is negatively modulated by transcription factors SNAI2 and TEAD1. Epithelial-specific factor ELF3 is a downstream effecter, and protein kinase C (PKC) links FZD5 to ELF3. ELF3 represses ZEB1 expression, further guarding against EMT. Moreover, FZD5 signaling requires its co-receptor LRP5 and WNT7B is a putative ligand for FZD5. FZD5 and ELF3 are associated with longer survival, whereas SNAI2 and TEAD1 are associated with shorter survival.Taken together, FZD5-ELF3 signaling blocks EMT, and plays a potential tumor-suppressing role in gastric cancer.Video Abstract The extracellular N-terminus contains a cysteine-rich domain (CRD) through which FZDs bind WNT ligands. The intracellular C-terminus binds the PDZ domain of Dishevelled (Dvl) and interacts with G proteins. Up to now, 10 FZDs have been identified in human. These receptors mediate β-catenin pathway and various β-catenin-independent pathways depending on cellular context. WNT/β-catenin pathway is oncogenic and involved in almost every aspect of tumor development. In addition, β-catenin-independent WNT/PCP and WNT/Cagression . In recegression –8, indicEpithelial-mesenchymal transition (EMT) in tumor cells endows them with enhanced motility thereby increasing their metastatic potentiality. This process is characterized by reduced expression of epithelial-related factors and increased expression of mesenchymal-related factors. Mechanistically, EMT is driven by some transcription factors such as SNAI1/2 and ZEB1/2. Studies have shown that FZD signalings induce tumor cell EMT and metastasis. FZD4, FZD7 and FZD10 promote EMT through β-catenin pathway in prostate, liver and breast cancer –11. FZD2In gastric cancer, FZD4 and FZD7 similarly promote EMT and metastasis by activating β-catenin pathway , 14. Genhttp://kmplot.com/analysis/) to assess the survival [Cancer Cell Line Encyclopedia (CCLE) database was interrogated for gene expression in 37 human gastric cancer cell lines. Correlation between two genes was analyzed by Pearson statistics. The Cancer Genome Atlas (TCGA) database was interrogated for GO pathway enrichment analysis in 375 gastric cancer tissues and 32 normal gastric tissues. GSE62254 database was interrogated in Kaplan Meier plotter website  secondary antibody (1:5000). Protein content was detected with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Primary antibodies for FZD5 (1:1000), SNAI2 (1:1000), LRP5 (1:1000) were purchased from Cell Signaling Technology. Primary antibody for ELF3 (1:1000) was provided by Thermo Scientific. Primary antibodies for TEAD1 (1:2000) and WNT7B (1:1000) were obtained from Abcam.Premix Ex Taq™ II (TaKaRa). Primers for genes and transcription factor binding sequences were listed in Table −ΔΔCT method.RNAiso Plus (Takara) was used to extract total RNAs, which were then reversely transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa)according to the instructions. Real-time PCR was performed using TB Green™ Cells were fixed with 4% paraformaldehyde, and incubated with PBS containing 0.1% Triton X-100. Slices were then incubated with primary antibodies for CDH1  and VIM , and subsequently incubated with Alexa 488 Donkey anti-Rabbit IgG secondary antibody . The fluorescence was visualized by a confocal microscope.Cells on the slides were fixed with 4% formaldehyde at room temperature for 10 min. Then the slides were washed with PBS 3 times, and incubated with 0.5% Triton X-100. TRITC-conjugated Phalloidin solution  was added on the slides incubated at room temperature for 30 min. DAPI was used to stain the nuclei. Finally, cell morphology was visualized by a confocal microscope.4 cells were added into the upper chamber. Culture medium with 10% FBS was added to the lower chamber. After 24 h, non-migrated cells were removed with a swab. The chambers were fixed with 4% paraformaldehyde for 15 min at room temperature, and incubated with 0.1% crystal violet for 30 min at 37℃. Cells were determined in 5 randomly selected microscope fields.Transwell chambers were used to determine cell migration ability. 2 × 10A ChIP assay kit  was used according to the manufacturer’s instructions. Briefly, Cells in 10 cm plates were fixed with 1% formaldehyde for 10 min at 37 °C. To shear the chromatin, cells were treated with 1 mM PMSF in SDS Lysis Buffer for 10 min at 4 °C, followed by cell sonication for 15 min at 4 °C. After a portion of the cross-linked chromatin was removed as input for the subsequent test, the remaining cell lysis was incubated with 1 μg primary antibody at 4 °C overnight. Then protein A + G agarose was added to precipitate the target protein recognized by the primary antibody for 1 h at 4 °C. IgG antibody was used as a negative control. The beads were then washed off and DNA was collected for subsequent Real-time PCR assays. The enrichment was indicated as % of input.500 µg protein was incubated with 2 µg primary antibody at 4 °C for 4 h. 20 µl Protein A/G PLUS Agarose beads (Santa Cruz Biotechnology) were added at 4 °C overnight. Then the agarose beads were washed to clear non-specifically bound proteins. The target protein was detected by Western blot.55 gastric cancer tissues were obtained from Shengjing Hospital China Medical University with the informed consent of the patients. Institutional Research Ethics Committee of China Medical University approved the use of these tissues for research purposes.Paraffin-embedded sections were deparaffinized and rehydrated. Sections were in turn added endogenous peroxidase blockers, normal non-immune animal serum, primary antibody, biotin–conjugated secondary antibody, streptomycin antibiotic-peroxidase solution, freshly prepared DAB. Then sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared and mounted. Primary antibodies for FZD5 (1:100) and TEAD1 1:200) was purchased from Abcam. Primary antibody for ELF3 (1:200) was provided by Thermo Scientific. Primary antibody for SNAI2 (1:200) were obtained from Cell Signaling Technology. H-Score system (range: 100–400) was used to assess the protein expression [0 was purt test or one way ANOVA was used to compare data among groups. Correlation analysis was performed using Pearson statistics. Survival was analyzed by Kaplan–Meier method. P value less that 0.05 was considered as significant.GraphPad Prism 7.0 was used for statistical analysis. Data were expressed as mean ± SD. Student's Interrogation of CCLE database revealed that FZD5 is positively correlated with epithelial-related factors CDH1 (E-cadherin), OCLN (Occludin), EPCAM and ELF3, while negatively correlated with mesenchymal-related factors VIM (Vimentin), SNAI2 (Slug), ZEB1 and ZEB2 in 37 gastric cancer cell lines (Fig. As FZD5 was shown to be inversely correlated with EMT transcription factor SNAI2 in mRNA level Fig. a, whetheYAP/TEAD was shown to interact with SNAI2 in modulating gene transcription , 22. TheAs shown in Fig. LRP5 is a co-receptor of FZDs. Interestingly, interrogation of CCLE database revealed that LRP5 is positively correlated with FZD5, CDH1, EPCAM and ELF3, while negatively correlated with VIM, SNAI2, ZEB1 and ZEB2 (Fig. Just like FZD5 knockdown, WNT7B knockdown in MKN45 cells inhibited CDH1 expression but induced VIM expression Fig. a, b. MorFZD5, ELF3, TEAD1 and SNAI2 expression in 55 gastric cancer tissues was detected by immunohistochemistry. Expression scoring showed that FZD5 is positively correlated with ELF3 while negatively correlated with TEAD1 and SNAI2 Fig. a. EMT poAmong 19 WNT ligands in human, WNT7A/B and WNT5A have been reported to bind to FZD5. WNT7A-FZD5 signaling promotes endometrial and ovarian cancer cell proliferation and growth through β-catenin pathway , 28. SimHowever, our study revealed that FZD5 is a putative suppressor in gastric cancer. Interrogation of databases showed an association of FZD5 with epithelial-like phenotype. Loss and gain of function studies further confirmed that FZD5 prevents cancer cell EMT. Prognosis analysis indicated that high FZD5 expression is correlated with longer survival period. Furthermore, our study showed that WNT7B is a putative ligand for FZD5 in gastric cancer. WNT7A/B seems to specifically bind to FZD5 among 10 FZDs . ActuallAs shown in our study, FZD5 is transcriptional inhibited by TEAD1-SNAI2 complex. Activation of tumor-suppressing Hippo pathway results in phosphorylation and degradation of Yap and Taz. When Yap and Taz are not phosphorylated, they translocate into the nucleus to bind transcription factor TEAD. Yap/Taz-TEAD signaling induces gastric cancer cell EMT, stemness, drug resistance and metastasis –37. IntrELF3 is a universal epithelial-limited transcription factor. Similar to FZD5, ELF3 has dual roles in human tumor. ELF3 induces breast epithelial cell malignant transformation , 42, andActivation of β-catenin pathway requires not only FZDs, but also their co-receptors LRP5/6 which is not involved in non-canonical β-catenin-independent pathway. A recent study reported that FZDs-LRP5/6 interaction can initiate β-catenin pathway in the absence of WNTs . SurprisIn summary, our study for the first time uncovers that FZD5-ELF3 signaling prevents EMT and is associated with favorable prognosis in gastric cancer. Therefore, FZD5 and ELF3 function as putative tumor suppressors in this type of cancer. Antibodies against FZD5 including ipafricept and vantictumab have been shown effective on certain cancer types . However


The Nb-rich blocky precipitates, MC carbides, MN nitrides, oxides, and fine γ’ was in all modified castings. Presence of other microstructural features, such as Ti-rich needles, eutectic γ-γ’ islands, small Al-rich and Cr-rich precipitates depended on the casting composition. The lowest solidus and liquidus temperatures were observed in superalloys with a high Al/Ti ratio. Consequently, in A7–A9 variants, the solidification range did not exceed 100 °C. In the A0 variant the difference between liquidus and solidus temperature was 138 °C. Hardness of all modified superalloys was at least 50% higher than for the remelted Inconel 740 (209 HV10). The chemical composition of standard Inconel 740 superalloy was modified by changes in the Al/Ti ratio  and addition of Ta . Remelted Inconel 740 (A0) and nine variants with various chemical compositions were fabricated by lost-wax casting. The microstructure, microsegregation, phase transformation temperatures, thermal expansion coefficients and hardness of the superalloys were investigated by scanning electron microscopy, energy dispersive X-ray spectroscopy, differential scanning calorimetry, dilatometry and Vickers measurements. Typical dendritic microstructure was revealed with microsegregation of the alloying elements. Segregation coefficient k In comparison to Nimonic 263, is has increased Cr and decreased Mo contents, respectively. Low content of Mo (around 0.5%) and lack of W ensure excellent coal ash resistance, which is one of the critical criteria for 700 °C. They favor the breakdown of protecting chromium oxide layers in contact with sulfate and chloride-containing ash deposits, thereby making some common alloys defenseless to pitting attack. The microstructure of Inconel 740 can consist of undesirable phases like silicon-rich G-phase and the DO24-ordered hexagonal η-Ni3Ti phase [Electricity is presently a key pillar of mankind’s living and economic development. Despite various methods of electricity production, a significant part of the energy sector is still based on coal. Forecasts predict that this state will continue for the next decades, despite the continuously repeated economic crises in the oil & gas branch. The factor that can successfully mitigate the effects of such crises is the continuous improvement of power unit efficiency. This has prompted the power sector to increase the pressure and temperature of their steam boilers to reduce COmissions ,2,3,4. Ds by 22% ,6,7. Coas by 22% ,9,10,11.s by 22% ,13,14. Ws by 22% ,16. Incos by 22% . To deveTi phase ,19,20,21Ti phase . The microstructure stability and strength of superalloys during long-term service depend strongly on the alloying elements and their concentrations. Alloying has always been the main design strategy for stabilizing phase compositions, microstructural constituents morphology, and mechanical properties like creep and fatigue resistance. Some investigations aimed at modifying the chemical composition of Inconel 740 to improve properties, including increasing γ’ phase stability and mechanical properties, counteraction of brittle phases’ formation and improvement of corrosion resistance, have been reported ,27,28,292O3, 53.0 SiO2, 1.0 Fe2O3, 1.6 ThO2, 0.1 N2O, 0.8 K2O; wt%). Stuccoing was carried out by immersing the slurry-coated model in a fluidized layer of sand. For the first and second layers, grain size 0.1–0.3 mm was used, whereas for the rest 0.5–1.0 mm. The established granulation of the quartz sands used was strictly observed. After coating of each layer, the model was air-dried, at 22 °C (±2 °C) and humidity 40–60%, so that the liquid components of the binder  evaporated. Drying time for individual layers was between 4 and 12 h, i.e., about 40 h in total. The wax models, covered with 7 layers of the ceramic, were dewaxed in a boilerclave for 20 min. at 140 °C and 4 atm. pressure. Then so-obtained shell molds were burned out at 850 °C for 6 h.The casting models and the gating system were made of saturated hydrocarbons (70%—paraffin), fatty acids (20%—stearin) and synthetic waxes (20%—polyethylene wax) mixture. The model mass was prepared in a special tank (equipped with an indirect heating system), where the individual ingredients were melted and mixed in the order: paraffin, stearin, polyethylene wax. Model mass with the required temperature, quality and consistency was obtained by cooling and thorough mixing. Wax models  were obtThe 3 kg “master heat” Inconel 740 ingots were melted in argon in a corundum crucible of a UltraMelt S10-SP furnace. Melting time was 25 min. and melt-homogenization was 10 min. Double deoxygenating was carried out manually with Ni15Mg compound granules, first after melting the superalloy and secondly, directly before pouring to the preheated mold (1250 °C). For each one 5 g of the compound was used. S-type thermocouples PtRh10-Pt  with 0.8 mm wire diameter were used for temperature measurements. After solidification of the molten alloy and ceramic knock-off, the upper part of each casting (75 mm) was cut off and scrapped and the bottom part (120 mm) was used for further investigation. The steps of the casting process are presented graphically in Remelted Inconel 740 (A0) became the base material for further chemical composition modifications, shown schematically in the 3) for light microscopy (LM) and electrochemically in 10% oxalic acid for scanning electron microscopic (SEM) observations. Using LM images the secondary dendrite arm spacing was measured in 20 various locations. SEM observations were on a Desktop XL instrument with CeB6 electron source and the accelerating voltage during SEM-BSE (backscattered electrons) observations and X-ray energy dispersive spectrometry (SEM-EDX) was 20 kV. Segregation coefficient ki = The as-cast specimens (10 × 10 × 10 mm) were cut off from the middle section of each casting and for microscopic examinations were mounted in resin, ground, polished and chemically etched in Aqua Regia  is interdependent with the local solidification time (St) and rate (v), temperature gradient (G) and cooling rate [The microstructure of the remelted Inconel 740 superalloy (A0) and all the variants with aluminium and tantalum (A1–A9) are shown in ing rate . The theing rate  postulating rate  reportedi = i is lower than 1 indicating that the element segregated to interdendritic spaces, while when it is greater than 1, the element is more concentrated in the dendritic regions. The most evenly distributed was Ni for which kNi values were in the range 0.93–0.97. For Cr, kCr higher than one was obtained in variants A0 to A6, while in A7–A9 the higher Al/Ti ratio did not exceed unity. Cobalt was the only alloying element whose concentration for each superalloy variant was higher in the dendrite arms (kCo < 1). The three highest kAl values were found for superalloys A7–A9 (Al/Ti > 3.0), which were characterized by kCr < 1. The elements that most strongly segregated to interdendritic spaces were Nb and Ti, for which the coefficients were 0.39 < kNb < 0.68 and 0.45 < kTi < 0.59. Similarly to Nb and Ti, Ta concentration was also higher in these spaces and kTa in each modified variant was below 1.Segregation of alloying elements occurring during solidification influenced substantially the large heterogeneity of microstructure. The coefficient k616Si7 formula. It forms on MC carbides or in close vicinity of M23C6 carbides. G-phase precipitates are undesirable because they lead to a decrease in creep resistance, and therefore their presence in Inconel 740H has been significantly limited. EDX measurements on the precipitates showed the mean chemical composition (at%): Ni-41.9 (±0.5), Si-20.9 (±1.2), Nb-18.4 (±1.7), Co-10.4 (±0.4), Ti-4.1 (±0.6), Cr-4.0 (±2.2). The average relation //Si was 7.5/17.4/7. These results were close to the general formula presented above. Evans [2 formula [SEM microstructures of the remelted Inconel 740 and EDX-spectra of detected precipitates are presented in e. Evans  found a  formula . Taking 24-ordered hexagonal Ni3Ti-η phase. The presence of η phase was also confirmed by Bouse [3Al (γ’) phase and promote Ni3Ti (η) phase. When the sum of Ti+Nb+Ta exceeds the Al concentration, the formation of η or other platelet phases is favored, either directly from the liquid, or by transformation from the eutectic γ-γ’ islands.In accord with by Bouse  in severAl being higher than 1 in variants A7–A9. Aluminum was uniformly distributed in the dendritic region and in the interdendritic spaces strongly Al-rich precipitates were present and the surrounding areas were slightly depleted in Al. In variants A0–A6 such areas were not present and kAl values were around 1, which is usually typical for superalloys. Also, the concentration of Ni was at a higher level there. Numerous fine precipitates within the interdendritic spaces consisted mainly of Cr. Increasing content of strong carbide formers led to formation of carbides in the following order M7C3→M23C6→M6C→MC [Cr was the lowest in variants A7–A9. Additionally, there were precipitates enriched in Nb and MC carbides consisting mainly of Nb, Ta, and Ti. Titanium nitride has also been detected inside the carbide. Modification of Inconel 740 by changing the Al/Ti ratio and introducing tantalum led to major changes in the morphology of primary precipitates . Dendrit6→M6C→MC . Taking 6→M6C→MC , the micChemical composition data of selected precipitates in the interdendritic space were based on EDX measurements at ten different locations in each casting. EDX maps revealed that generally three elements formed the MC carbides. Their contents are presented as ratios: Nb/Ti, Nb/Ta, and Ti/Ta in 0 as a function of temperature in the range of 25–1200 °C. At 1200 °C, ∆L/L0 in each of the modified alloys was clearly higher than in the reference A0, for which it was 21.764 × 10−3. The three highest ∆L/L0 values, 24.25 × 10−3, 24.15 × 10−3 and 23.49 × 10−3, were observed in variants A4, A9 and A8, respectively. Up to about 800 °C, the ∆L/L0 for each of the alloys was increasing linearly, and more rapidly as temperature increased. The gradual slope of A8 and A9 curves around 1000 °C can be related to some secondary reprecipitation from the solid solution. A similar slope was observed in model Ni-Ta-Al-C alloys by Bala [γ) and γ’ (aγ’) in several superalloys is only similar for lower temperatures. At higher temperatures, aγ starts to increase more rapidly with increasing temperature than aγ’. The coefficient of thermal expansion of A0 variant in the range 25–1200 °C was 1.86 × 10−5 K−1. Among the modified alloys, the highest CTE was 2.07 × 10−5 K−1(A4), while the lowest was 1.93 × 10−5 K−1 (A2). All obtained values were shown in  by Bala , who pos by Bala  reportedST) and liquidus (LT) temperatures of remelted Inconel 740 estimated by DSC were 1230 °C and 1368 °C, which gave the solidification range 138 °C. The enthalpy of fusion calculated in accordance withS are the lowest. Slight differences in the relationships were observed for the liquidus temperature. Three almost parallel zones related to the Al/Ti ratio are clearly evident on L below 1300 °C) liquidus temperatures was 60 °C. The influence of the chemical composition (Al/Ti ratio and Ta content) on the solidification range shown on ST and LT, but the tantalum concentration C, TiN, MgO were found. Moreover, phases exhibiting features typical for G-phase and η were detected.-In all modified castings, the Nb-rich blocky-precipitates, C carbides, TiN nitrides, oxides, and fine γ’ were observed. In A1–A3 and A6 the Ti-rich needles (probably η phase) were detected, whereas in A4 and A5 variants: eutectic γ-γ’ islands. Variants with the highest Al/Ti ratios had additionally the Al-rich and Cr-rich precipitates.-With an increase of Ta concentration in each superalloy group  the Nb/Ti, Nb/Ta and Ti/Ta concentration ratios in carbides decrease gradually.-In the A0 variant, solidus and liquidus temperatures were higher than in the modified alloys, respectively 1230 °C and 1368 °C. The lowest temperatures observed were in the variants with a high Al/Ti ratio, which also resulted in a narrow solidification ranges, not exceeding 100 °C. With the decrease in Al/Ti, the range of crystallization was wider, even more than 130 °C. These results indicated a much stronger effect of Al than Ta on these phase transformation temperatures.-−5 K−1, while the highest in A4, 2.07 × 10−5 K−1.All modified variants had higher coefficients of thermal expansion in the temperature range 25–1200 °C. In A0 variant it was 1.86 × 10-Hardness of the reference as-cast Inconel 740 was 209 HV10. By modification of the chemical composition this value increased at least by 50%. The highest hardness value was in A8 variant, 423HV10.Nine newly-developed superalloys based on Inconel 740 were fabricated by investment casting in an induction furnace in argon. The remelted Inconel 740 (A0 variant) was modified by various Al/Ti ratios  and Ta concentrations . From this work, the following conclusions can be drawn:


Nkuku® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the  Aphthovirus genus in the family Picornaviridae  have not been isolated for FMDV serotype A, SAT1, and SAT3. The scFvs resulting from the biopanning were investigated in virus neutralization assays in the pursuit of epitope identification and for their prospective use as FMDV diagnostic reagents in an ELISA.Baby hamster kidney (BHK) strain 21 clone 13 cells (ATCC CCL-10), used for virus propagation and SAT1 and SAT3 serotype neutralization assays, were maintained in Glasgow minimum essential medium , supplemented with 10% (v/v) fetal bovine serum , 1 × antibiotic-antimycotic solution (Invitrogen), 1 mM L-glutamine (Invitrogen), and 10% (v/v) tryptose phosphate broth  cells used for A22 virus propagation and virus neutralization tests, were maintained in Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum  and 1 × antibiotic-antimycotic solution (Invitrogen). The Mycl-9E10 hybridoma (ECACC 85102202) was cultured in protein-free hybridoma medium (Invitrogen).The SAT1/KNP/196/91 and SAT3/KNP/10/90 FMDVs used for the biopanning, originated from buffalo in the Kruger National Park (KNP) in South Africa, isolated during 1991 and 1990, respectively . Also usNkuku® phage display library was performed as described by van Wyngaardt et al. (® Maxisorp), after being coated overnight with purified virus (30 μg/ml) were blocked with 1 × PBS containing 2% (w/v) milk powder (Elite) and incubated with the Nkuku® library phage particles (1012-1013 transducing particles). Exponentially growing Escherichia coli TG1 cells  were infected with eluted phage-displayed scFvs that had bound to the specific viruses before plating on TYE plates  supplemented with 2% (w/v) glucose and 100 μg/ml ampicillin. Subsequent to overnight incubation, the bacteria were collected and the phagemids rescued by the addition of M13KO7 helper phage. Infected bacterial cells were incubated overnight in 2 × TY medium  containing 100 μg/ml ampicillin and 25 μg/ml kanamycin. Phages displaying scFvs were precipitated from the cell-free culture supernatant with one-fifth of the original culture volume of 20% (w/v) PEG-8000 in NaCl and were then suspended in 1 × PBS for use in the next selection round. A total of three such selection rounds were performed. The input and output phages from each selection round was titered to monitor enrichment. The outputs of each consecutive selection round was tested in a polyclonal ELISA. Single clones from the third selection round was tested as soluble scFvs for specific binding to SAT1/KNP/196/91, SAT3/KNP/10/90, or A22.Selection of virus-specific scFvs from the t et al.  and Oppet et al. . Briefly®) overnight at 4°C. To confirm the specificity of the phage-displayed scFvs to the respective viruses, 1 × PBS containing 2% milk powder (Elite) was used as a blocking reagent and negative control. Bound PEG-precipitated phage-displayed scFvs, produced at each selection round, were detected with the MAb B62-FE2  and horseradish peroxidase-conjugated polyclonal rabbit anti-mouse IgG . After a final wash step, substrate/chromogen solution consisting of 4 mM 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) in substrate buffer  and 0.015% H2O2 was added for the colormetric reaction. Following 10 min incubation at room temperature, the color reaction was stopped with 1 M H2SO4 and the absorbance values were recorded at an absorbance of 450 nm (A450nm).van Wyngaardt et al.  and Oppe®) containing 2 x TY medium supplemented with 100 μg/ml ampicillin and 2% (w/v) glucose. The bacteria were grown overnight with shaking at 30°C. Using a 96-well inoculation device (Sigma-Aldrich: Cat. No. R-2508), bacterial cells were transferred from the overnight plate to a second plate containing 150 μl of fresh medium per well-followed by incubation for 2.5 h at 37°C with shaking. Subsequently, 50 μl of medium that contained 2 × 109 of the M13K07 helper phage was added to each well and the plate incubated for 30 min at 37°C without shaking. Thereafter, plates were centrifuged at 600 × g for 10 min, the supernatant fractions were removed and replaced with 150 μl of 2 × TY medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin prior to incubation overnight at 30°C with shaking. Following centrifugation at 600 × g for 10 min to pellet bacterial cells, the supernatant fractions, which contained the phage-displayed scFvs were removed and mixed 1:1 with 1 × PBS containing 4% (w/v) casein and 0.2% (v/v) Tween-20 prior to undergoing ELISA testing as described for the polyclonal phage ELISA above.Following the third round of panning, monoclonal phage antibodies were screened by randomly selecting individual clones from the titration plates and inoculating it into a 96-well tissue-culture plate  as per the manufacturer's instructions. The OP52 forward primer (5′-CCCTCATAGTTAGCGTAACG-3′) and M13 reverse primer (5′-CAGGAAACAGCTATGAC-3′), as well as the ABI PRISM™ Big Dye™ Terminator Cycling Ready Reaction Kit v.3.0 (Applied Biosystems) was used to sequence the single clones ]. The clones were inoculated into 2 x TY medium containing 100 μg/ml ampicillin and 20% (w/v) glucose and phagemid DNA was isolated with a QIAprepe clones . An ABI e clones  and SequGlycerol stocks (150 μl) of selected phage clones were inoculated in 90 ml of 2 × TY medium  containing 100 μg/ml ampicillin and 2% (w/v) glucose and incubated overnight at 30 °C with shaking. A 1:10 dilution of the overnight culture was prepared in 800 ml of fresh 2 × TY medium containing 100 μg/ml ampicillin and 2% (w/v) glucose and incubation, with shaking, continued for a further 8 h after which the bacterial cells were pelleted at 4,000 × g for 30 min. All traces of glucose-containing 2 × TY media was removed and the bacterial pellet resuspended in 1 L of 2 × TY media containing 100 μg/ml ampicillin and 1 mM IPTG and incubated overnight at 30°C with shaking. The expressed soluble scFv was harvested by pelleting the bacterial cells at 4,000 × g for 30 min.3, 0.02% sodium azide; pH 8.0) and treated with 0.01% (v/v) of 100 mg/ml lysozyme for 30 min at 30°C. Freshly prepared 200 mM phenylmethylsulfonyl fluoride [0.01% (v/v)] in isopropanol was added to the bacterial suspension and the suspension mixed by inverting. The bacterial suspension was sonicated for 3 min (30 s pulses with 30 s pauses), where after, the bacterial pellet was collected by centrifugation at 15,000 × g for 30 min and the clear lysate filtered through 0.8 and 0.45 μm filters, respectively. The anti-myc-Sepharose column coupled with 143 mg of 9E10 Mab  was washed with TSA buffer before the clear lysate (containing soluble scFvs) was loaded through the column via a peristaltic pump. The column was washed with TSA buffer until the spectrophotometer reading at absorbance A280nm fell below 0.3. Soluble scFvs were eluted from the column with elution buffer  and fractions collected. Peak fractions were pooled and the scFvs dialyzed , for 48 h at 4°C, in 2 L PBS pH 7.4.The bacterial pellets were resuspended in 50 ml of TSA buffer  sucrose and 1 × PBS containing 2% (w/v) milk powder as negative controls.50) of A22 . Similarly, BHK-21 cells were used to determine the virus titers for SAT1/KNP/196/91 and SAT3/KNP/10/90. The resulting virus titers were used to calculate the dilutions subsequently used in the virus neutralization test (VNT).IB-RS-2 cells were used to determine the 50% tissue culture infective dose (TCID2 after purified scFvs at a concentration between 0.03 and 0.23 mg/ml were added neat to appropriate wells. A control plate without soluble scFvs was included. BHK-21 and IB-RS-2 cells supplemented with 1% (v/v) FCS and antibiotics , with a cell count of 0.3 × 106 cells/ml for both cell lines, were subsequently added to the respective microtiter plates. Incubation of microtiter plates then occurred for 72 h at 37°C and fixation and staining with a methylene blue-formaldehyde stain to allow for inspection of the cytopathic effect, which was scored as a measure of neutralization.Virus dilutions containing ~500, 50, and 5 infectious particles were prepared in the appropriate cell medium (RPMI for IB-RS-2 cells and GMEM for BHK-21 cells) and were applied in triplicate wells across a microtiter plate and diluted two-fold down the plate. Virus was incubated for 1 h at 37 °C in an atmosphere of 5% COTo generate virus neutralization escape mutants, the viruses  were passaged under scFv pressure as described by Crowther et al. . Equal vVirus escape mutants were then characterized by sequencing the Leader- P1-2A coding region (ca. 3 kb) of the virus genome. RNA extraction was performed using the QIAmp viral RNA kit (Qiagen). cDNA was synthesized with SuperScript III first strand synthesis kit (Invitrogen), using the genome specific primer WDA 5′-GAAGGGCCCAGGGTTGGACTC-3′.2 and 2 μM of each dNTP. Using a thermocycler  after initial denaturation at 94°C of 2 min, the reactions were subjected to 35 cycles of 94°C for 20 s, 55°C for 20 s and 68°C for 4 min with a final cycle of 68°C for 7 min to complete the synthesis of all strands.The Leader-P1-2A coding region of the escape viruses were amplified using the Expand Long Template PCR System (Roche) and forward primer NCR1 5′-TACCAAGCGACACTCGGGATCT-3′ and reverse primer WDA 5′-GAAGGGCCCAGGGTTGGACTC-3′. Briefly, each 50 μl PCR reaction mixture consisted of 3 μl of the first strand cDNA reaction mixture, 0.3 μM of each oligonucleotide, 2.5 U of Expand Long Template DNA polymerase, 1 × Expand buffer, 0.75 mM MgClTo determine the nucleotide sequence of the gel-purified amplicons, 0.16 μM of the appropriate oligonucleotide  and the 450nm) of ca. one was obtained. The virus dilution of 1:8 was chosen for the scFv ELISA as this was the highest dilution where an A450nm ~1  was obtained for the viruses tested. The purified, neat scFvs  were used to coat 96-well Maxisorp immunoplates (Nunc®) overnight at 4 °C, following which, ELISA plates were washed four times with wash buffer [1 × PBS containing 0.05% (v/v) Tween 20]. As a blocking reagent and negative control, 2% milk powder in 1 × PBS was used. Diluted PEG concentrated virus (1:8) was added to the scFv coated plates and incubated for 1 h at 37°C. Following incubation and washing, serotype specific guinea pig antiserum  was added (working dilution for SAT1 and SAT2 was 1:100 and SAT3 whilst for A it was 1:50) and plates were again incubated for 1 h at 37°C and then washed. The conjugate  diluted at 1:80, was added to respective microtiter plate wells, followed by 1 h at 37°C incubation and washing. The ELISA colorimetric reaction followed using substrate-chromogen solution, consisting of 4 mM 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) in substrate buffer  0.015% (v/v) H2O2. Following 10 min incubation at room temperature, the colorimetric reaction was stopped with 1.25M H2SO4. The A450nm was determined using a Labsystems Multiskan Plus photometer (Thermo Fisher Scientific). The samples were tested in duplicate wells and the absorbance calculated as an average of the two values for each sample. A positive ELISA result was calculated as two-fold the A450nm value of the average negative control.The SAT1 and SAT3 soluble scFvs were tested in an indirect ELISA against a panel of viruses  to deter®) were coated with either SAT1, SAT2, SAT3, or A specific rabbit antiserum. A 1:8 dilution of PEG concentrated viruses (®) and incubated for 1 h at 37°C. Following a wash step with wash buffer i.e., 1 × PBS containing 0.05% (v/v) Tween 20, undiluted scFvs  were added and ELISA plates incubated for 1 h at 37°C. Microtiter plates were washed and the soluble scFvs that bound to the FMD antigen were detected with the anti-c-Myc antibody clone 9E10 (Sigma-Aldrich) and 1: 1,000 dilution of horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse IgG . The negative control contained 2% milk powder (Elite) in 1 × PBS instead of the scFvs. The substrate/chromogen solution and A450nm determination was performed as described above and a positive A450nm value was considered greater-than or equal to two-fold the average negative control value. The test samples were tested in duplicate and the absorbance calculated as an average of the two values for each test sample. The LPBE was adapted and essentially carried out in conjunction as a comparison of performance of the scFv detecting ELISA and was undertaken as described in the Office International des Epizooties  produced A450nm results of 0.13, 0.10, and 0.06 for SAT1/KNP/196/91, SAT3/KNP/10/90, and A22, respectively. An increase of at least eight-fold in the absorbance values after three pannings compared to the absorbance of a pre-panning aliquot of the Nkuku® library proved enrichment for phage displayed antibodies for all three FMD viruses , A22scFv2 (0.23 mg/ml), and A22scFv8 (0.18 mg/ml), were able to neutralize A22 in vitro. The neutralization titers (TCID50) are indicated in The ability of the soluble scFv's against SAT1, SAT3, and A22 to neutralize the SAT1/KNP/196/91, SAT3/KNP/10/90, or A22 viruses, respectively, FMDV A22 was serially passaged in the presence of soluble A22scFv1, A22scFv2, and A22scFv8 to select viruses from the A22 quasispecies population that escape neutralization by the soluble scFvs. Thus, the A22 viruses that escaped neutralization by soluble scFvs A22scFv1, A22scFv2, and A22scFv8 were designated scFv resistant virus (SRV) 1, 2, and 3, respectively. Following four consecutive passages under scFv pressure, the P1 nucleotide, and aa sequences were determined for SRV1, SRV2, and SRV3. Comparative analysis of the aa sequences of the SRVs compared to the parental A22 sequence indicated that SRV1 exhibited one aa substitution i.e., from a non-polar proline (Pro) to a polar serine (Ser) change at VP1 aa position 149 i.e., RGD+3 (Pro149➔Ser) . InteresThe A22 virus was neutralized by three soluble scFvs and SRVs for A22, which showed a potential binding site for two of the scFvs in the GH-loop of the VP1 protein. The three CDRs of the H-chain of A22scFv1 and A22scFv8 are identical while the L-chain of A22scFv1 and A22scFv8 displayed different sequences . Solublen = 11) viruses whilst soluble SAT3scFv1 and SAT3scFv2 was tested against a panel of SAT3 (n = 12) viruses  (450nm 0.31), SAT3/KNP/1/03 (A450nm 0.31), and SAT3/ZIM/11/94 (A450nm 0.40) viruses indicating this scFv may recognize the same epitope on the virion for these viruses (SAT3scFv1 and SAT3scFv2 soluble scFvs did not react efficiently with the SAT3 viruses when applied as a capturing reagent and displayed A0nm 0.6) . SAT3scF viruses . AdditioOverall, the ELISA results using the scFvs as capturing reagents indicate that the soluble SAT1scFv1 was able to successfully bind to the polystyrene ELISA plate and react to viruses within the SAT1 serotype. The SAT3scFv1 and SAT3scFv2 exhibited no or borderline reactivity with the SAT3 viruses tested. The low signals may be attributed to the conformational changes of the scFvs when binding to the ELISA plate. Furthermore, aa differences in the viral proteins may result in the variable ELISA signals observed.The soluble scFvs, SAT1scFv1, SAT3scFv1, and SAT3scFv2, were also applied as detecting antibodies in a sandwich ELISA. FMD virus was captured by polyclonal rabbit antiserum and the soluble scFvs was used to detect the 146S virus particles using the panel in 450nm >1.4 was observed for SAT1/SAR/9/03, SAT1/KNP/196/91, and SAT1/NAM/272/98 viruses and (ii) A450nm absorbance values of ≥0.4, ≤ 0.82 for the remaining eight SAT1 viruses in the panel  of 0.76 and 0.84 were observed for SAT3/KNP/10/90 and SAT3/KNP/6/08, respectively when SAT3scFv1 was the detecting antibody in the ELISA. All other A450nm values were below 0.26 for the SAT3 viruses tested with SAT3scFv1 and SAT3scFv2  can be found in the article/supplementary material.MC: majority of the experimental work, troubleshooting, result and data analysis, and manuscript composition and editing. PO: scFv expertise and training, experimental work, troubleshooting, result and data analysis, manuscript editing, and research grant acquisition. LR: serotype A experimental work, troubleshooting, result and data analysis, and manuscript editing. JFe and WW: scFv expertise advisor, Nkuku library, and manuscript editing. JFr: scFv column development, scFv purification training and expertise/advisor, and manuscript editing. ER: research grant acquisition, PI on grant at PIADC, project advisor, and manuscript editing. FM: research grant acquisition, PI on grant at ARC, project advisor, scFv expertise, and manuscript editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lactobacillus rhamnosus GG (LGG) ATCC 53103 was used to treat BALB/c mice. After LGG intervention, both low and high LGG doses were shown to improve the observed OTU, Chao1, ACE, and Shannon indices, while the Simpson index decreased, demonstrating that LGG can promote intestinal microbiota abundance and diversity. Furthermore, LGG treatment increased the abundances of intestinal Firmicutes, Bacteroides and Actinomycetes while reducing that of Proteobacteria. In addition to its effect on gut the microbiota, LGG could also regulate the host immune system. In the present study, we showed that LGG could affect the percentage of CD3+ T lymphocytes in the spleens (SPLs), mesenteric lymph nodes (MLNs), Peyer’s patches (PPs) and lamina propria lymphocytes (LPLs) of mice, including total CD3+ T, CD3+CD4+ T, and CD3+CD8+ T lymphocytes. Furthermore, LGG could effectively increase the expression of Th1-type cytokines (IFN-γ) and Th2 cytokines (IL-4) in CD4+ T cells, indicating that the proportion of Th1 and Th2 cells in mice with LGG treatment was in a high equilibrium state compared to the control group. In addition, the IFN-γ/IL-4 ratio was greater than 1 in mice with LGG intervention, suggesting that LGG tends to mediate the Th1 immune response. The results of the present study also showed that LGG upregulated the expression of IL-17 in CD4+ T cells and regulated the percentage of CD4+CD25+Foxp3+ Treg cells in various secondary immunological organs, indicating that LGG may promote the balance of Th-17 and Treg cells.Lactic acid bacteria (LAB) are the primary genera of the intestinal flora and have many probiotic functions. In the present study,  Lactobacillus rhamnosus LS-8 and Lactobacillus crustorum MN047 supplementation possessed the anti-obesity effect on the HFFD fed mice by alleviating inflammatory response and regulating gut microbiota, which further suggested that these two probiotics can be considered as an alternative dietary supplement in combination with the preventive and therapeutic strategies against obesity and related complications is the most frequently used strain. LGG strain is a Gram-positive bacteria isolated from the human digestive system for the first time in 1985 by Sherwood Gorbach and Barry Goldin. LGG regulate the intestinal flora through the mechanism of “competitive rejection,” where the colonization of pathogens in the intestinal mucosa is hindered by increasing competition for adhesion sites and nutrients, production of antibacterial compounds , ompounds , regulatompounds , maintaiompounds , as wellompounds . Previouacterium . LGG grar damage . LGG supude mice .Lactobacillus can regulate both innate and adaptive immunity, playing a role in disease prevention and treatment in the host through immune stimulation and regulation. The effect of Lactobacillus on innate immunity is primarily achieved by enhancing the phagocytic ability of the monocyte-phagocytic system. Studies have shown that Lactobacillus can enhance the phagocytic activity of mouse peritoneal macrophages and play a regulatory role in the immune system (e system . In addie system .Lactobacillus johnsonii (NCC 533) or Lactobacillus paracasei (NCC 2461) induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that LAB can activate mucosal B-cell responses  abundance of analysis, we found enrichment of intestinal flora pathways in LLGG group, including Biosynthesis of amino acids, Carbon metabolism, ABC transporters, and Ribosome pathways, etc .+ T cells in the spleens (SPLs) of mice in the control, LLGG and HLGG groups were 32.9 ± 0.68%, 38.52 ± 0.88%, and 37.86 ± 0.88%, respectively. Compared to the percentage of CD3+ T cells in the SPLs of mice in the control group, a higher percentage was observed in those of LLGG group  and HLGG group . The percentages of CD3+ T cells in the mesenteric lymph nodes (MLNs) of mice in the control, LLGG and HLGG groups were 74.84 ± 0.71%, 71.72 ± 0.27%, and 77.48 ± 0.63%, respectively. The percentage of CD3+ T cells in the MLNs was lower in the LLGG group  and higher in the HLGG group  than that observed in the control group. The percentages of CD3+ T cells in lamina propria lymphocytes (LPLs) in the control, LLGG and HLGG groups were 29.04 ± 0.89%, 32.30 ± 0.38%, and 33.38 ± 0.82%, respectively. Compared to the percentage of CD3+ T cells observed in the LPLs of mice from the control group, higher percentages were observed in the LLGG group  and HLGG group . The percentages of CD3+ T cells in Peyer’s patches (PPs) of mice in the control, LLGG and HLGG groups were 30.04 ± 0.50%, 27.82 ± 1.42%, and 35.86 ± 1.41%, respectively. The percentage of CD3+ T cells in PPs from mice in the HLGG group was higher than that observed in the control group  and LLGG group   .+CD4+ T cells are important immune cells. After 7 LGG interventions, the percentages of CD3+ CD4+ T cells among CD3+ T lymphocytes in the SPL in the control and HLGG groups were 50.30 ± 0.72% and 52.48 ± 0.41%, respectively. The percentage of CD3+ CD4+ T cells among CD3+ T lymphocytes in the SPLs of mice in the HLGG group was higher than that observed in the control group . The percentages of CD3+ CD4+ T cells among CD3+ T lymphocytes in MLNs from mice in the control, LLGG and HLGG groups were 71.76 ± 0.24%, 72.62 ± 0.27% and 70.96 ± 0.25%, respectively. The percentages of CD3+ CD4+ T cells among CD3+ T lymphocytes in PPs from the control, LLGG and HLGG groups were 62.48 ± 0.46%, 68.30 ± 1.60% and 69.08 ± 1.29%, respectively. Compared to the percentage of CD3+ CD4+ T cells observed among CD3+ T lymphocytes in PPs in the control group, higher percentages were observed in the LLGG group  and HLGG group . The percentages of CD3+ CD4+ T cells among CD3+ T lymphocytes in the LPLs from the control, LLGG and HLGG groups were 49.76 ± 1.23%, 53.30 ± 0.84% and 57.20 ± 0.94%, respectively. The percentage of CD3+ CD4+ T cells among CD3+ T lymphocytes in the LPLs from the HLGG group was higher than that observed in the control group  and LLGG group , while the percentage observed in the LLGG group was also higher than that measured in the control group   .+ CD8+ T cells among CD3+ T lymphocytes in SPLs from the control, LLGG and HLGG groups was not significantly different. The percentages of CD3+ CD8+ T cells among CD3+ T lymphocytes in the MLNs from the control, LLGG and HLGG groups were 25.22 ± 0.17%, 23.86 ± 0.28%, and 26.16 ± 0.23%, respectively. The percentage of CD3+ CD8+ T cells among CD3+ T lymphocytes in MLNs from the HLGG group was higher than that observed in the control group  and LLGG group , while the percentage in the LLGG group was lower than that observed in the control group . The percentages of CD3+ CD8+ T cells among CD3+ T lymphocytes in PPs from the control, LLGG and HLGG groups were 12.08 ± 0.26%, 11.01 ± 0.30%, and 14.96 ± 0.57%, respectively. The percentage of CD3+ CD8+ T cells among CD3+ T lymphocytes in PPs from the HLGG group was higher than that observed in the control group  and LLGG group , while the percentage in the LLGG group was lower than that observed in the control group . The percentages of CD3+ CD8+ T cells among CD3+ T lymphocytes in the LPLs from the control, LLGG and HLGG groups were 15.28 ± 0.83%, 18.42 ± 0.73%, and 18.56 ± 0.52%, respectively. Compared to the percentage of CD3+ CD8+ T cells observed among CD3+ T lymphocytes in the LPLs from the control group, a higher percentage was observed in the LLGG group  and HLGG group   .+ T, CD3+ CD4+ T and CD3+ CD8+ T cells in peripheral lymphoid organs (SPLs), intestinal secondary lymphoid organs  and intestinal tissue (LP). In the LLGG group, a significant increase the percentage of CD3+ T cells in the SPLs, CD3+ CD4+ T cells in the MLNs and PPs, and CD3+, CD3+CD4+, and CD3+CD8+ T cells in the LPLs was observed, while a significant decrease in the percentage of CD3+ T and CD3+ CD8+ T cells was observed in the MLNs and PPs. Thus, a high dose of LGG had a strong effect on the percentage of all subsets of T cells, which can better improve the immune function of mice.In summary, both low-dose and high-dose LGG intervention significantly affected the percentage of CD3BALB/c mice at 6 weeks of age received 7 high- or low-dose LGG treatments on alternate days, and a PBS control group was also established. SPLs, MLNs, PPs, and LP of the three groups of mice were isolated after the interventions to prepare single-cell suspensions. PMA, ionomycin, and BFA were added to the single-cell suspensions. Lymphocytes were collected 6 h later, and IFN-γ was labeled with a specific fluorescent antibody and detected by flow cytometry. IFN-γ, also known as type II interferon, is one of the most important types of Th1 cytokines.+CD4+ IFN-γ+ Th1 cells in the SPLs in the control, LLGG and HLGG groups were 2.14 ± 0.08%, 3.142 ± 0.20%, and 3.054 ± 0.14%, respectively. Compared to the percentage of CD3+CD4+ IFN-γ+ Th1 cells in the SPLs from the control group, a higher percentage was observed in the LLGG group  and HLGG group   and HLGG group . The percentage of CD3+CD4+ IFN-γ+ Th1 cells in the MLNs from the HLGG group was also higher than that observed in the LLGG group   . The per 0.0351) . Comparep < 0.05, p = 0.0091) and HLGG group . The concentration of serum IFN-γ in the HLGG group was also higher than that observed in the LLGG group   .+ T cells to differentiate into Th2 cells. After 7 LGG interventions, the percentages of CD3+CD4+IL-4+ Th2 cells in the SPL in the control, LLGG and HLGG groups were 2.142 ± 0.0955%, 2.730 ± 0.218%, and 2.660 ± 0.14%, respectively. Compared to the percentage of CD3+CD4+IL-4+ Th2 cells observed in the SPLs from the control group, a higher percentage was observed in the LLGG group  and HLGG group   and HLGG group  (IL-4 molecules are primarily secreted by Th2 cells and can promote B cells to proliferate and induce CD4 0.0155) . The per 0.0076) . Comparep < 0.05, p = 0.0472) and HLGG group . The concentration of serum IL-4 in the HLGG group was also higher than that observed in the LLGG group   .+ T cells in the SPLs and MLNs was greater than 1  and HLGG group   and HLGG group   . The per 0.0012) . No sign+CD25+Foxp3+ Treg cells in the SPLs from the control, LLGG and HLGG groups were 11.23 ± 0.05%, 11.62 ± 0.13%, and 11.16 ± 0.09%, respectively. The mice in the LLGG group had a higher percentage of CD4+CD25+Foxp3+ Treg cells in the SPL than those in the control group  and HLGG group   , while a higher percentage was observed in the HLGG group . The percentage of CD4+CD25+Foxp3+ Treg cells in PPs from the HLGG group was significantly higher than that observed in the LLGG group   . The per 0.0492) . The per 0.0001) . CompareIn summary, LGG could affect the differentiation of Th1, Th2, Th17, and Treg cells in peripheral lymphoid organs (SPLs) and intestinal secondary lymphoid tissues (MLNs and PPs) of mice. LGG could promote the differentiation of Th0 cells into Th1, Th2, and Th17 cells and enhance the immunity of mice. The effect of the high-dose LGG treatment was stronger than that of the low-dose treatment. The LLGG treatment could significantly increase the proportion of Treg cells in the SPL and reduce that observed in PPs, while the HLGG treatment could significantly increase the proportion of Treg cells in PPs and reduce that observed in the MLNs, thereby regulating the balance between tolerance and immunity.Lactobacillus can colonize the intestinal tract, regulate the intestinal flora, increase the number of the dominant intestinal flora, improve intestinal health, and effectively prevent the infection of pathogens. Based on high-throughput sequencing analysis, LGG ATCC53103 fed to gnotobiotic pigs was shown to maintain intestinal balance and increase the dominant intestinal flora, especially to maintain the basal level of enterococci and effectively resist rotavirus infection , suggesting that LGG tends to mediate the Th1 immune response. In addition, the results of the present study showed that LGG upregulated the expression of IL-17 in CD4+ T cells. An increase in IL-17 promotes the ability of the host to resist infection by bacteria, mycobacteria and fungi  enhances IFN-γ production and inhibits IL-4 production in mitogen activated mouse and human spleen T cells, while high concentrations (≥1 × 106 CFU/mL) inhibit mitogen-induced T cell proliferation /ml.LGG ATCC 53103 was grown in De Man, Rogosa, and Sharpe (MRS) broth for 12 h at 37°C. After culturing overnight, the bacteria were inoculated 1:100 in fresh MRS broth and grown under anaerobic conditions until reaching the mid-log phase. Then, the colonies were counted, and the cell density was adjusted to 5 × 103, 104, 105, 106, 107, 108, and 109CFU. The mice in the high-dose LGG group (HLGG) were intragastrically administered 109CFU every other day for 7 total treatments. One week after the LGG interventions (on the 21st day), the 45 from the three groups were sacrificed.Forty-five 6-week-old female BALB/c mice were randomly divided into 3 groups, with 15 mice in each group. The mice in the normal control group (Control) were intragastrically administered PBS (200 μL) every other day for 7 total treatments. The mice in the low-dose LGG group (LLGG) were intragastrically administered LGG every other day for 7 total treatments, with intervention doses of 10® High-Fidelity PCR Master Mix (New England Biolabs), 2 μM of forward and reverse primers and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, elongation at 72°C for 30 s, and finally 72°C for 5 min. Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with Qiagen Gel Extraction Kit . Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit  following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.Total genome DNA from samples was extracted using CTAB method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/μL using sterile water. 16S rRNA genes of distinct regions (16S V3-V4) were amplified used specific primer (341F: 5′-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACNNGGGTATCTAAT-3′) with the barcode. All PCR reactions were carried out with 15 μL of Phusion1 .After sequencing, paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH (V1.2.7)1 . Quality1  accordin1 . Then, the fat was removed, and the small intestine was cut longitudinally. Subsequently, the intestinal tissue was rinsed with precooled PBS (without Ca2+ and Mg2+) at 4°C until clean, after which it was transversely cut into intestinal segments 0.5∼1 cm in length. The intestinal segments were then placed in 5 mL of intraepithelial lymphocyte (IEL) separation solution  and incubated at a constant temperature (37°C) with shaking (200 r/min) for 15 min. Subsequently, the intestinal segments were passed through a 200-mesh nylon filter and then added to 5 mL of IEL separation solution, after which the above filtration procedure was repeated after shaking at 37°C for 15 min. The remaining intestinal segments were placed in 5 mL of lamina propria (LP) digestion solution , 100 kU/L DNase I, and 5% FBS in RPMI 1640 medium). After shaking (200 r/min) at a constant temperature (37°C) for 45 min, the remaining intestinal segments were filtered with a 300-mesh aseptic filter, and the solid residue was discarded, with the filtrate collected in a 15-mL aseptic tube. After centrifugation at 4°C and 400 × g for 10 min, the supernatant was discarded to collect the LP cells. Then, 4 mL of an 80% isotonic Percoll solution was placed at the bottom of a 15 mL centrifuge tube, and the LP cell pellets were resuspended in 7 mL of a 40% isotonic Percoll solution. The resuspended LP cell pellets were fully mixed, pipetted onto the 80% isotonic Percoll solution, and then centrifuged at 20°C and 2,300 rpm for 20 min. Subsequently, the upper liquid layer was discarded, and the cells between two liquid levels were transferred to a new 15-mL centrifuge tube, after which PBS (without Ca2+ and Mg2+) was added to a volume of 15 mL. After centrifugation at 4°C and 2,000 rpm for 8 min, the supernatant was discarded. Then, the remaining cell pellets were washed twice with PBS (without Ca2+ and Mg2+), resuspended and counted.A 10-cm section of small intestine harboring Peyer’s patches was placed in cold PBS , PerCP-CD3 (clone: 17A2), phycoerythrin (PE)-CD3 (clone: 17A2), fluorescein isothiocyanate (FITC)-CD4 (clone: GK1.5), allophycocyanin (APC)-CD8 (clone: 53-6.7), PE-CD25 (clone: PC61), PE-IFN-γ (clone: XMG1.2), APC-IL-4 (clone: 11B11), PE-Cy7-IL-17 (clone: TC11-18H10), and APC-Foxp3 (clone: MF23). All antibodies were purchased from BD Biosciences .6 single cell suspensions were seeded into the 48-well cell culture plate, and 0.5 mL of complete medium was added to each well. Each well was incubated with 20 ng/mL of phorbol 12-myristate 13-acetate  and 1 μg/mL ionomycin (Sigma) at 37°C for 4 h, after which GolgiStop (BD Biosciences) was added and the samples were incubated for 2 h. Then, the cells were resuspended in 100 μL PBS (without Ca2+ and Mg2+) and incubated with 10 μL of specific CD3 and CD4 monoclonal fluorescent antibodies at room temperature for 30 min away from light. Subsequently, the cells were washed twice with the proper amount of PBS (without Ca2+ and Mg2+) and centrifuged at 4°C and 2,000 rpm for 5 min, after which the supernatant was discarded. Then, the cell pellets were mixed well with 250 μL of fixation/permeabilization solution and incubated at 4°C for 20 min. One milliliter of 1 × BD Perm/WashTM buffer was directly added to each tube, and after centrifugation at 4°C and 2,000 rpm for 5 min, the cells were washed twice, and resuspended in a volume of 100 μL. Subsequently, 10 μL monoclonal fluorescent antibodies against IFN-γ, IL-4, and IL-17 was added to each sample tube and incubated at 4°C for 30 min after mixing. Each tube was then washed twice with the proper amount of PBS (without Ca2+ and Mg2+), leaving 300 μL of buffer to resuspend the cells. Subsequently, the cell suspension was passed through a 300-mesh nylon filter membrane into the flow and detected on an LSR-FORTESA flow cytometer (BD Biosciences).For intracellular cytokine staining, 1∼2 × 10+CD25+Foxp3+ Treg staining, 1 × 106 single cell suspensions were incubated with 10 μL of specific monoclonal fluorescent antibodies against CD4 and CD25 and at room temperature without light for 30 min. Then, 1 mL of freshly prepared 1 × Fix/Perm Buffer working solution was directly added to each tube. The tubes were mixed by shaking for 3 s and then incubated at 4°C without light for 40 min. The fixed and permeabilized cells were resuspended in 1 mL of 1 × Perm/Wash Buffer, centrifuged at 4°C and 350 × g for 6 min, and then the supernatant was discarded. The cell pellets were resuspended in 2 mL 1 × Perm/Wash Buffer and centrifuged at 4°C and 350 × g for 6 min, and the supernatant was discarded. The cells were then resuspended in 100 μL of 1 × Perm/Wash Buffer, mixed with an appropriate amount of specific anti-Foxp3 antibody, vortexed for 10 s to mix well and then incubated at 4°C without light for 40 min. Each tube was then washed twice with 2 mL of 1 × Perm/Wash Buffer and centrifuged at 4°C and 350 × g for 6 min, and the supernatant was discarded. The cell pellets were then resuspended in 300 μL of PBS (without Ca2+ and Mg2+), passed through a 300-mesh nylon filter membrane into a tube and detected on an LSR-FORTESA flow cytometer (BD Biosciences). The data were analyzed using FlowJo 9.3 .For CD4The IFN-γ and IL-4 levels in serum were detected using a commercial ELISA kit (R&D SYSTEMS) following the manufacturer’s instructions. Briefly, the standard was diluted and loaded according to the manufacturer’s instructions, and the blank and sample wells were appropriately prepared. On the precoated plate, 50 μL of the diluted sample solution was added to the well of the experimental sample to be tested. The sample was added to the bottom of the plate well without touching the well wall, after which the samples were gently shaken to mix well. The sealed plate was ten incubated at 37°C for 30 min. The sealing film was then carefully removed, the was liquid discarded, and each well was filled with wash buffer and incubated for 30 s and before discarding the liquid, with the process repeated 5 times. Then, 50 μL of enzyme-labeled reagent was added to each well, except for the blank well, incubated at 37°C for 30 min and then the wells were washed five times. Subsequently, 50 μL of chromogenic agents A and B were added to each well, mixed by gently by shaking and then incubated at 37°C for 15 min without light. Then, 50 μL of terminator solution was added to each well to terminate the reaction. The plate was placed into a microplate reader, the blank well was adjusted to zero, and the absorbance of each well was measured at 450 nm.p < 0.05.The data were analyzed using GraphPad Prism 5.0. Student’s t-test was used to compare two groups, and one-way ANOVA was used to analyze more than two groups. Differences were considered significant when PRJNA 675996).The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethics Committee of Jilin Agricultural University.CS, XC, GY, and CW conceived and designed the experiments and wrote the manuscript. CS, XY, YL, MC, and RW performed the experiments. MC, YJ, DZ, JW, and HY performed high through sequencing analysis. CS, YZ, WY, HH, and LY performed the flow cytometry analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the  The availability of large amounts of proteins that have potential biomedical applications is often limited in the initial steps of research. Particularly demanding is the setup of in vitro screening programs to investigate protein-protein interactions, ligand binding and other properties to evaluate their potential as pharmaceutical agents ,2. This O-methyltransferase (COMT) inhibitor approved for Parkinson’s disease, is also an efficient TTR tetramer stabilizer and has been repurposed for FAP [Human transthyretin (hTTR) is a 55 kDa homotetrameric serum protein with a main role in transporting thyroid hormones T4 and T3) and retinol through binding to the retinol-binding protein (RBP)  and T3 a,7,8, TTR for FAP ,22 and f for FAP . Recentl for FAP  is now i for FAP . Another for FAP .hTTR has also shown to have a neuroprotective role in Alzheimer disease (AD). AD is a multifactorial neurodegenerative disorder  characteEschericchia coli is often hampered by low production yields. Few studies have reported to achieve high cell density cultures with moderate recombinant TTR productivity. The first protocols produced secreted protein into the culture medium to facilitate purification but with low production yields, in the range of 5 mg/L in flask cultures [N-terminus methionine was added for recombinant expression in E. coli cells as compared to the native sequence of mature human TTR after signal peptide processing with production yields of purified protein in the range of 150 mg/L [Studies aimed at finding TTR ligands acting as amyloidogenesis inhibitors for therapeutic intervention in TTR-associated amyloidotic polyneuropathies or acting as molecular chaperones to enhance the TTR:Aβ interaction and improve the neuroprotective effect in AD do require large amounts of hTTR protein to conduct in vitro screening assays. TTR expression in recombinant cultures ,38. Intrcultures , but thecultures . We repo150 mg/L . Here, wN-terminus methionine was added in order to express the closest form to mature human TTR [The gene coding for human TTR was cloned in a pET38b vector for high protein expression where no tags and just an uman TTR . Here, wE. coli transformants and cells were grown at 37 °C until an OD600 of 0.6 before induction  or 2× YT medium supplemented with kanamycin and IPTG induction at 37 °C . When monduction A. IPTG iE. coli BL21(DE3) cells was used [To adapt the protocol for expression in a bioreactor, an optimized defined medium (DM) for was used . A fed-bHigher cell densities in the fed-batch step were obtained with an enriched feeding medium composed of DM with 25 g/L glucose supplemented with tryptone (8 g/L) and yeast extract (5 g/L). The fed-batch started when the initial glucose was consumed and IPTG at a final 1 mM concentration was added at the beginning of the fed-batch in a single addition. The fed-batch lasted 4.5 h before harvesting the cells. After purification (see below), the final production yield was 660 mg/L of culture for the Y78F mutant hTTR. The final protocol described in the Materials and Methods section was reproducible for the wt and different TTR mutants, with average production yield of purified proteins in the range of 200–660 mg/L of culture, depending on the protein variant. This is the highest reported production yield for recombinantly expressed hTTR enabling the application of screening protocols that demand high amounts of protein.4)2SO4). TTR precipitated in the third fractions (between 55 and 85%). The precipitate was dissolved in Tris buffer and dialyzed against the same buffer. The first chromatographic step was anion exchange chromatography with Q-Sepharose at pH 7.6. At this high pH, about 2 pH units above the TTR isoelectric point , the pH may vary from 7.2 to 8.0 with no effect on final protein yields. In the standardized protocol (see methods) TTR was eluted with a gradient from 0.1 to 0.5 M NaCl in Tris buffer pH 7.6. The combined TTR-containing fractions were extensively dialyzed and lyophilized. The lyophilization step was introduced after checking that it did not affect the properties of the final protein  since it simplifies the concentration of the diluted protein before gel filtration chromatography. The lyophilized protein was dissolved in a small volume of Tris buffer pH 7.6, 0.1 M NaCl and loaded into a Superdex 75 gel filtration column. The eluted protein was dialyzed against water and lyophilized for storage and dissolved in the corresponding buffer before use.The cells harvested by centrifugation (either from flask or bioreactor cultures) were lysed by sonication or with a cell homogenizer (in the case of large cultures). After centrifugation, the supernatant was subjected to fractional ammonium sulfate precipitation  was assessed by SDS-PAGE electrophoresis, MALDI-TOF mass spectrometry, and a fibrillogenesis assay.w/v) SDS and heating the samples at 100 °C for 2 min. An example of purified protein after the expression and purification protocol is shown in Native TTR is an homotetramer that appears as a dissociated monomer with traces of dimer under standard SDS-PAGE conditions with a loading buffer containing 2% (S-Sulfo), S-glycinylcysteinylation (S-CysGly), S-cysteinylation (S-Cys) and S-glutathionylation (S-GSH) as the most prevalent forms [E. coli, the produced proteins, both wt and Y78F mutant, were modified at Cys-10. S-GSH). However, the Y78F mutant produces mainly the glutathionated form and the S-Cys, S-Sulfo and cysteic acid forms as minor components. Treatment of Y78F hTTR with DTT reduces the S-GSH form to free TTR as shown in Native hTTR occurs in vivo as a very heterogeneous protein due to post-translational modifications (PTM) at Cys-10, the single Cys residue in the protein sequence. It has been reported that only around 10–15% of the circulating TTR in plasma remains unmodified at Cys-10, finding S-sulfonation  protein is about 1.25-fold more amyloidogenic than the reduced protein (free SH). When fibrillogenesis of S-GSH and reduced proteins was monitored up to 72 h, both protein forms reach the same amount of final aggregates/fibrils  monitor acid-induced aggregation and fibrillation, commonly by lowering the pH to 4.2–4.4 for wt and low amyloidogenic mutants, or higher pH values (6–7) for highly amyloidogenic mutants. These methods include turbidimetric assays and thioflavin T binding assays (reviewed in ). Here, /fibrils B. In conThe high expression yields of recombinant hTTR, either wt, highly amyloidogenic variants or clinically relevant mutants obtained with our optimized protocol (200–660 mg/L of culture depending on the mutant) as homogeneous proteins have enabled their application in screening programs for the evaluation of libraries of TTR amyloidogenesis inhibitors as well as the evaluation of small-molecule chaperones enhancing the interaction between TTR and Amyloid-β (Aβ) peptides.50, concentration of inhibitor at which the initial rate of fibril formation is one-half than that without inhibitor, and RA(%), percent reduction of fibril formation rate at high inhibitor concentration relative to the rate in the absence of inhibitor. The initial protocol reported in [50 values in the range of 3–15 μM and for RA(%) values in the range of 60–100% when using different protein batch productions.Based on the acid-induced properties of hTTR, this assay evaluates the ability of small molecules to inhibit fibrillogenesis/aggregation in a dose-dependent manner to quantify two relevant inhibition parameters: ICorted in  has beenorted in ,48,49,50Inspired by the previous assay, we developed a rapid and simple high-throughput assay to screen for small-molecules that may act as chaperones of the TTR:Aβ interaction. The assay monitors Aβ(12-28) aggregation in the presence of both wt TTR and a small molecule compound (SMC). The small-molecule chaperones form ternary soluble complexes TTR/Aβ/SMC that prevent Aβ aggregation. The method uses the shorter Aβ(12-28) sequence, a cheaper model system for aggregation by Aβ(1-40). The test is carried out in 96-well plates that are UV monitored for turbidity for 6 h. E. coli BL21(DE3) cells harboring the corresponding plasmids with kanamycin resistance as selection marker. Final protocols for protein expression were:Recombinant wt and Y78F mutant TTR were expressed from phTTR.wt-I and phTTR.Y78F-I plasmids based on a pET38b(+)  vector as reported . Both wtE. coli cells and grown overnight at 37 °C. 100 μL of the initial culture were used to inoculate 50 mL of 2xYT+ 100 μg/mL Kan at 37 °C until an optical density (OD600) of ~4. Finally, 1.3 L of 2xYT+ 100 μg/mL Kan were inoculated with 5 mL of the previous culture in a 2 L Erlenmeyer flash with deflectors for improved aeration. Cells were grown at 37 °C with shaking at 200× rpm until an OD600 of 0.6 (about 6 h). IPTG was added to a final 1 mM concentration and the culture incubated at 30 °C for 14–16 h. Cell were harvested by centrifugation at 4 °C, 10,000× rpm for 10 min and directly used for protein purification.A total of 2 mL LB (or 2× YT) medium supplemented with 100 μg/mL kanamycin (Kan) was inoculated with a single colony from an LB-agar plate of freshly transformed 2HPO4, 2.65 g/L KH2PO4, 2.04 g/L NaCl, 4.1 g/L (NH4)SO4, 0.026 g/L FeCl3, 0.5 g/L MgSO4·7H2O, 0.01 g/L thiamine, 25 g/L glucose and 2.86 mL/L Trace Elements Solution composed of 0.04 g/L AlCl3·6H2O, 0.16 g/L CoCl2·6 H2O, 0.01 g/L H3BO3, 0.01 g/L NiCl2·7H2O, 0.87 g/L ZnSO4·7H2O, 1.55 g/L CuSO4·5H2O, 1.42 g/L MnCl2·4H2O, 0.02 g/L NaMoO4, 1.44 CaCl2·2H2O. A 10 L bioreactor  containing 4 L of DM supplemented with 100 mg/L Kan was inoculated with 50 mL of freshly prepared culture in DM medium at OD600 of 0.6. The batch culture was grown at 37 °C with increasing agitation (300 to 1000 rpm) to maintain the dO2 at 5–10%. When glucose was consumed and the dissolved oxygen increased, the fed-batch was initiated with a feeding solution of DM supplemented with 100 mg/L kanamycin, 8 g/L tryptone and 5 g/L yeast extract, the temperature was set at 30 °C and the culture induced with IPTG  at the beginning of the fed-batch step. During all the culture the pH was maintained at 7.0 with NH4OH (30% stock solution). During the fed-batch, agitation was maintained at 1000 rpm, and pO2 at 5–10% by supplying additional oxygen if dO2 < 5% or adding feeding solution if dO2 > 5%. At 4.5 h after IPTG induction, cells were harvested by centrifugation at 4 °C, 10,000 rpm for 15 min and the pellet directly used for protein purification.The defined medium (DM) is composed of 13.23 g/L KAll volumes in the purification steps are given for processing the cell pellet coming from 1L culture. The cell pellet was resuspended in 250 mL lysis buffer , and lysed with a cell disrupter at 20 kpsi . Viscosity of the homogenate was reduced by applying short sonication cycles before centrifugation at 4 °C, 12,000× rpm for 30 min to collect the supernatant.4)2SO4 up to 40, 55 and 85%. The precipitate fraction after the last step (55 to 85% fraction) contained most of the TTR protein (as judged by SDS-PAGE). The precipitate was resuspended in 50 mL of buffer A , and dialyzed (3×) against buffer A at 4 °C using a MWCO 6–8 kDa, 40 mm Cellu.Sep Regenerated Cellulose Tubular Membranes .(i) Ammonium sulphate precipitation. Three precipitation steps at 4°C were performed by addition of (NH(ii) Anion exchange chromatography. The dialyzed solution was loaded to a XK 26/40 column (120 mL) with Q-Sepharose High Performance resin using an ÄKTA FPLC System . Buffer A was used for column conditioning (at 1 mL/min) and protein loading (at 2 mL/min). Elution (1 mL/min) was done with a NaCl linear gradient (0.1 M to 0.5 M NaCl in Buffer A). Fractions were collected and analyzed by SDS-PAGE. All fractions containing TTR were combined and dialyzed against deionized water at 4 °C (MWCO 6–8 kDa) three times for 8 h. The final solution was lyophilized.(iii) Gel filtration chromatography. The lyophilized sample was dissolved in 4 mL of Buffer A and loaded at 2 mL/min flow rate onto a XK 26/100 column (440 mL) with Superdex 75 prep Grade resin using an ÄKTA FPLC System . Protein elution was performed with Buffer A at 0.3 mL/min and eluted fractions were collected and analyzed by SDS-PAGE. Fractions containing TTR were combined and dialyzed against deionized water at 4 °C  and lyophilized for storage at −20 °C.2HPO4, 0.1 M KCl, pH 7.6 buffer at 4 °C.A 4 mg/mL TTR solution in 20 mM Tris·HCl, 0.1 M NaCl, pH 7.6 was treated with 1 mM DTT during 1h at room temperature. Completion of the reduction reaction was checked by MALDI-TOF MS. The reduced solution was dialyzed against 20 mM Kv/v) acetonitrile:water, 0.1% trifluoroacetic acid (TFA) and a protein solution in 30:70 (v/v) acetonitrile:water, 0.1% TFA were prepared and mixed in a 1:1 ratio. A total of 1 µL of the previous mixture was deposited into a polished stainless-steel target  and allowed to dry. Then, 1 µL of SA matrix solution was deposited into the sample and allowed to dry. The same procedure was followed for the Protein Standard Calibration I solution (Bruker) used for Calibration. The target was introduced in a Autoflex MALDI-TOF (Bruker), spectra were acquired in lineal mode  and processed by Flex Analysis (Bruker).A saturated solution of sinapinic acid (SA) as matrix in 30:70 (2O (1:1). Incubation buffer was added up to a volume of 100 μL. The 96-well plate was introduced into the thermostated microplate reader for 30 min at 37 °C, with orbital shaking for 15 s every min. Fibril formation was then induced by addition of 100 μL of dilution buffer to each well. The 96-well plate was placed again into the microplate reader and incubated at 37 °C with shaking (15 s every minute) during 1.5 h. Absorbance at 340 nm was monitored at each minute. All assays were run in triplicate.TTR stock solution: 4 mg/mL in 20 mM phosphate, 100 mM KCl, pH 7.6. Incubation buffer: 10 mM phosphate, 100 mM KCl, 1 mM EDTA, pH 7.6. Dilution buffer: 400 mM sodium acetate, 100 mM KCl, 1 mM EDTA, pH 4.2. The assay was performed in 96-well microplates. 20 μL of TTR stock solution were added into each well. The final DMSO content of each well was adjusted up to 5%  with DMSO:H2O (1:1). Incubation buffer was added up to a volume of 200 μL. The vials were sealed and incubated at 37 °C for 30 min. Afterwards, fibril formation was induced by addition of 200 μL of dilution buffer to each vial. After soft mixing, 200 μL of each vial were placed into a well in a 96-well plate and absorbance at 340 nm was measured . After measurement, the 200 μL were placed back into its corresponding HPLC vial, which was sealed again and placed at 37 °C. Measurements were performed at 1, 3, 6, 21, 30, 45, 54, 69 and 72 h following the same procedure. All assays were run per duplicate.For end-point assays, the same procedure was performed in sealed HPLC vials in order to avoid evaporation. A total of 40 μL of TTR stock solution was added to each vial. The final DMSO content was then adjusted up to 5% with DMSO:H2O (1:1) was added to give final concentrations ranging from 0 to 40 μM and DMSO final content of 5% . Incubation buffer (60 μL) was then added up to a volume of 100 μL. The plate was incubated at 37 °C in a thermostated microplate reader with orbital shaking 15 s every minute for 30 min. 100 μL of dilution buffer were dispensed to each well, and the mixture was incubated at 37 °C with shaking (15 s every min) in the microplate reader. Absorbance at 340 nm was monitored for 1.5 h at 1 min intervals. Data were collected and analyzed using Microsoft Excel software. All assays were done in duplicate. From the initial rates (Abs vs. time) at different inhibitor concentrations, the inhibition parameters IC50 and RA (%) were calculated as reported [Protein stock solution, incubation buffer and dilution buffer were prepared as above. Protocol for one inhibitor: 20 μL of hTTR Y78F stock was dispensed into seven wells of a 96-well microplate. A total of 20 μL of inhibitor solutions in DMSO:Hreported ,49,50,51The following stock solutions were used: Buffer A: 25 mM HEPES buffer, 10 mM glycine, pH 7.4. Protein (wt hTTR) stock: 9.5 mg/mL (170 µM) in 25 mM HEPES buffer, 10 mM glycine, pH 7.4 and 5% DMSO  was prepared in the absence of salt (buffer A). Aβ(12–28) stock: 0.4 mg/mL (200 µM) in 25 mM HEPES buffer, 10 mM glycine, pH 7.4 and 5% DMSO . For the small-molecule compound, a first 10 mM solution in DMSO was prepared and the final stock was prepared by mixing 50 µL of the previous DMSO solution with 950 µL of buffer A . First, the small-molecule compound and TTR complex was formed. To this end, 60 µL of TTR stock was dispensed into the wells of a 96-well microplate. A total of 40 µL of small-molecule stock was added to give final concentrations of 100 µM. The plate was introduced in the thermostated microplate reader  and incubated for 1 h at 37 °C with orbital shaking 15 s every 30 min. Then, 100 μL of Aβ solution was added to the well to give 100 μM ligand:50 μM TTR:100 μM Aβ final concentrations. The plate was incubated at 37 °C in the microplate reader with orbital shaking 15 s every minute for 30 min. The absorbance at 340 nm was monitored for 6 h at 30 min intervals. Data were collected and analyzed using MS Excel software . All assays were done in duplicate. From the different absorbance readings between sample and controls, the percent reduction of aggregates formation (RA%) in the presence and absence the small-molecule compound/TTR complex were determined.S-glutathionated form. When comparing the S-glutathionated and reduced Y78F proteins, amyloidogenicity (as determined by a turbidimetry assay) is little affected by this post-translational modification. The inter-batch reproducibility of expression and protein quality has allowed the implementation of screening assays for the evaluation of libraries of TTR amyloidogenesis inhibitors, as well as the evaluation of small-molecule chaperones of the TTR:Aβ interaction.The optimized expression and purification protocol for recombinant hTTR production (wt and amyloidogenic mutants) yields up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. Whereas the recombinant wt protein is mainly unmodified at Cys10, the highly amyloidogenic Y78F variant renders mainly the


Compared to the widely studied (Fe78Si9B13)100Zr0 (Zr0) amorphous ribbon for degradation, with increasing cZr (Zr atomic content), the as-spun Zr0, Zr0.5 and Zr1 amorphous ribbons have gradually increased degradation rate of MB solution. According to The as spun amorphous (Fe Nowadays, organic dyes wastewater is commonly produced in the industrial production of dyestuff, textiles, paper and plastics. This organic dyes wastewater contains carcinogenic, teratogenic and biological toxic substances, which will cause serious pollution to the environment ,2,3. ThuAmorphous alloys  have excellent properties such as high strength and corrosion resistance due to their long-range disordered atomic structures, and the importance of fundamental scientific and engineering application potential is attracting rising attention ,17,18. A78Si9B13 and Fe73.5Si13.5B9Cu1Nb3 amorphous ribbons, showed higher degradation efficiency when degrading cibacron brilliant red 3B-A, methyl blue and methyl orange dyes by Fenton-like reaction , which proved that Fe-based amorphous ribbons have better degradation performance of organic dyes than other kinds of amorphous alloys [76Si9B12Y3 powder to degrade methyl orange dye, which was 1000 times more reactive than industrial iron powder [Among Fe-, Cu-, Al- and Mg-based amorphous alloys used to degrade organic dyes wastewater, Fe-based alloys have attracted the most attention because of their high degradation efficiency, low cost and good reusability. The frequently used Fe-based alloys for degradation are FeSiB systems, and FeSiB alloys with other elements. Jia et al., using the prepared Fes alloys ,38. Xie n powder .78Si9B13)99.5Zr0.5 (Zr0.5) and (Fe78Si9B13)99Zr1 (Zr1) amorphous ribbons, the methylene blue (MB) dyes degradation by Fenton-like reactions is reported for the first time, and is compared with Fe78Si9B13 (Zr0) amorphous ribbon. The addition of trace Zr element effectively adjusts the interatomic force of Zr0 amorphous ribbon, which makes the atoms on the surface of amorphous ribbon participate in Fenton-like reaction more easily, and with the increase of Zr element content, it is easier to form developed 3D nano-porous acicular structure on the ribbon surface, thus increasing the specific surface area of reaction and improving the degradation rate. The effects of initial pH and H2O2 concentration on the degradation efficiency of MB using the Zr0, Zr0.5 and Zr1 amorphous ribbons during Fenton-like reactions are investigated. This work not only provides a new routine for the remediation of organic dyes wastewater, but also extends the application range of Fe-based amorphous/glassy alloys.In this paper, using (Fe78Si9B13)100Zr0 , (Fe78Si9B13)99.5Zr0.5  and (Fe78Si9B13)99Zr1  were prepared by arc melting of pre-alloyed Fe78Si9B13 ingots and high-purity Zr (99.99 wt.%) in an arc melting furnace , which was vacuumed to 5 × 10−3 Pa first and then filled with purified argon (99.999%). The ribbons with thickness of ~30 μm and width of ~2.5 mm were prepared by single copper roller melt-spinning  system. The roller/wheel speed was 42 m·s−1. The amorphous ribbons were cut into 5 cm long strips for degradation tests. Commercially available methylene blue , Hydrochloric acid , Sodium hydroxide  and Hydrogen peroxide  are used in the experiment.Alloy ingots with a nominal composition of (FeThe amorphous structure of the as-spun ribbons (in a stripe-like shape) was investigated by X-ray diffraction  Technology Co., Ltd., Beijing, China) with Cu-Kα radiation and transmission electron microscopy . The amorphous character of the ribbon samples was also confirmed by differential scanning calorimetry  at a heating rate of 20 K/min. The surface morphology of the as-spun and reacted ribbons was observed by scanning electron microscope  equipped with an energy dispersive X-ray spectrometer (EDS).−1) in 250 mL beakers. A certain mass of ribbons (0.5 g L−1) and H2O2 (1 mM) was placed to the MB solution, stirring at a fixed speed (300 r min−1) during the degradation process, and the constant temperature (298 K) of the MB solution was maintained with a water bath. The pH (pH = 3) of the MB solution was adjusted using 12 mol L−1 HCl, as well as 1 M NaOH. At time intervals, a 3 mL solution was extracted with a syringe and filtered with a 0.45 μm membrane, and the concentration of MB solution was monitored in real-time with UV-Vis spectrophotometer (UV-4802) to obtain the absorbance spectrum of the solution.Preparing MB solution in 500 mL volumetric flask with deionized water (DW), then pour 100 mL MB solution . The three-electrode cell was used for measurement, the saturated calomel electrode (SCE) was used as reference electrode, platinum was used as counter electrode and as-spun ribbon was used as working electrode. When the open-circuit potential stabilizes, the polarization curve was recorded at the potential scanning speed of 1 mV s−1. EIS was performed in static states, with scanning frequencies from 100 kHz to 0.01 Hz and the amplitude of ± 10 mV.The polarization curves and electrochemical impedance spectra (EIS) were measured using an electrochemical measuring instrument  in the 20 mL DW or MB solutions 100Zr0 (Zr0), (Fe78Si9B13)99.5Zr0.5 (Zr0.5) and (Fe78Si9B13)99Zr1 (Zr1) ribbons. The XRD curves of as-spun Zr0, Zr0.5 and Zr1 ribbons have only a typical diffuse scattering peak at 2θmax = 44.45°, 44.32° and 44.14° respectively, indicating that the as-spun Zr0, Zr0.5 and Zr1 ribbons owns a fully amorphous structure. With the increase of Zr content, the diffuse scattering peak moves to a low degree. The mean neighboring atomic distance increases with Zr content due to the large atom size of Zr element [TP1 and TP2, a melting peak temperature TP3 in the heating scan, respectively. The TP2-TP1  of as-spun Zr0, Zr0.5 and Zr1 ribbons are 18, 41 and 68 K, respectively. Not only are the two crystallization peak temperatures TP1 and TP2 of as-spun Zr0.5 and Zr1 ribbons higher than that of as-spun Zr0 ribbon, but also their crystallization peak temperature range of TP2-TP1 higher than that of as-spun Zr0 ribbon, indicating that the as-spun Zr0.5 and Zr1 ribbons have better amorphous stability and formability. The melting peak temperature TP3 of as-spun Zr0.5 and Zr1 ribbons was lower than that of as-spun Zr0 ribbon, indicating that the addition of Zr element can reduce the bonding force between atoms in amorphous ribbons, thus have a lower melting peak temperature. element . Figure In order to further characterize the microstructure of the as-spun Zr0, Zr0.5 and Zr1 ribbons, we conducted TEM investigations, which are shown in tr the absorption peak at 654 nm gradually decayed, indicating that the chromophore groups of MB disappeared gradually. In the first 9 min, the auxochrome groups react more quickly with Fe-based ribbons than the chromophore groups. The degradation kinetics is usually described by the pseudo-first-order equation as follows [k is the reaction rate constant (min−1), C0 is the initial concentration of MB solution (mg L−1), tr is the reaction time (min), and tC is the instant concentration of MB solution (mg L−1) at tr. In this work, the ln (C0t/C) − tr curves are shown in the inset of k of as-spun Zr0.5 and Zr1 ribbons are 0.22 min–1 and 0.24 min–1, which is larger than 0.19 min–1 for as-spun Zr0 ribbon. Here, the fit goodness values R2 of Zr0, Zr0.5 and Zr1 ribbons are 0.97, 0.98 and 0.97, respectively. Thus, the as-spun Zr0.5 and Zr1 ribbons bear a higher degradation performance for MB solution compared with as-spun Zr0 ribbon. follows :Ct = C0 In order to deeply understand the MB solution degradation mechanism with the Zr0, Zr0.5 and Zr1 amorphous ribbons, we test to study the structural evolution of the ribbon surfaces during the Fenton-like reaction process. The SEM images on the surface of the as-spun and reacted Zr0, Zr0.5 and Zr1 ribbons are displayed in θmax = 44.56°, 44.47° and 44.39° respectively, indicating that they still own a fully amorphous structure. Here, the different peak positions of the reacted ribbons are higher than the as-spun ribbons. Moreover, the 2θmax deviation Δ2θmax values of Zr0, Zr0.5 and Zr1 ribbons are 0.11°, 0.15° and 0.25°, respectively, showing a high tendency with increasing cZr. After reacting with MB solution for 15 min, the Zr0, Zr0.5 and Zr1 ribbons have a decreased cFe, and the cZr has remained basically unchanged. Moreover, the decrease of Fe element on the surface of Zr1 ribbon is higher than that of Zr0 and Zr0.5 ribbons, indicating that the high degradation rate of Zr1 ribbon is due to a large amount of Fe element participating in the Fenton-like reaction. After degradation, the cO on Zr0, Zr0.5 and Zr1 ribbon surface increases, indicating that the degradation process involves the oxidation of the ribbons.The XRD patterns of Zr0, Zr0.5 and Zr1 ribbons  are shown in Ecorr) of the Zr0.5 and Zr1 ribbons are −0.75 and −0.71 V (icorr) of the Zr0.5 and Zr1 ribbons are 5.83 × 10−6 and 3.30 × 10−6 A·cm−2, lower than the Zr0 ribbon (8.23 × 10−6 A cm−2). In MB solution, the Ecorr of the Zr0.5 and Zr1 ribbons are −0.60 and −0.54 V . The above data from polarization curves indicate that the Zr0.5 and Zr1 ribbons have better corrosion resistance than the Zr0 ribbon in DW and MB solution.The polarization curves and electrochemical impedance spectra (EIS) of the as-spun Zr0, Zr0.5 and Zr1 ribbons in DW and MB solution  for the Zr0, Zr0.5 and Zr1 ribbons are 1.47 × 10−5, 1.07 × 10−4 and 6.54 × 10−4 in DW, respectively; while they are 1.05 × 10−5, 6.77 × 10−6 and 7.71 × 10−6 in MB solution, respectively. In the equivalent circuit, the phase element (CPE) Q is defined as [Q is the resistance, j is the imaginary unit, w is the frequency, n is the coefficient of CPE and Y0 is the admittance.In both DW and MB solution c,d, the fined as :Q = (jw)Rs (solution resistance) of the Zr0, Zr0.5 and Zr1 ribbons in MB solution is lower than that in DW, which may be due to the conductive H+ in MB solution. The Rf (resistance of passivation film) and Ra  of the Zr0, Zr0.5 and Zr1 ribbons in DW are higher than those in MB solution respectively, due to a certain amount of H+ and methylene blue molecules in the MB solution. The Rtotal  of the Zr0, Zr0.5 and Zr1 ribbons in DW is higher than that in MB solution, and the Rtotal of Zr0.5 and Zr1 ribbons are higher than that of Zr0 ribbon in either DW or MB solution. Thus, the EIS results are also in good agreement with the results of polarization curves  of as-spun Zr0, Zr0.5 and Zr1 ribbons is lower than that at pH = 3 (+ in the solution is too high (H+ + Fe0 → Fe2+ + H2 ↑). This hydrogen evolution reaction generates a large amount of Fe2+, which may consume •OH and lower the oxidation capability of the MB solution (Fe2+ + •OH → OH− + Fe3+). As pH > 3, the η of the MB solution decreases with the increasing pH, as there must be enough H+ in the solution to carry out Fenton-like reactions. When the pH increases to 4, this MB solution hardly degrades before 7 min, and then it degrades slowly. As pH > 5 the η of MB solution is nearly zero.The working pH range of the Fenton-like reaction using the as-spun Zr0, Zr0.5 and Zr1 ribbons for the MB solution degradation has been studied, and keep other reaction conditions constant: t pH = 3 d. This m2O2 controls the rate of •OH generation in the Fenton-like reactions. The effect of the concentration on the degradation process of MB solution using as-spun Zr0, Zr0.5 and Zr1 ribbons was investigated, as shown in 2O2, including 0, 0.5, 1, 5, 10, 30 and 50 mM, are added to MB solution, and other reaction conditions constant: T = 298 K, pH = 3, ribbon dosage = 0.5 g L−1 and CMB = 100 mg L−1. It is proved that H2O2 is necessary to degrade MB solution, because the η is very low without adding H2O2 . The oxidation potential of generated radical •HO2 is much lower than that of •OH, which slows the degradation rate of MB solution.The concentration of Hing H2O2 d. When t2O2 concentrations. The surface of the as-spun Zr1 ribbon is relatively smooth  degradation, the pathway of this Fenton-like reaction can be drawn, as shown schematically in TP1 and TP2 of the Zr1 ribbon is higher than that of the Zr0 ribbon, which can make the network in the glassy matrix more stable. Hence, the ligament is more easily formed in the reacted Zr1 ribbon. Moreover, FexOy is not compatible with ligaments and leave the channel active, which helps iron atoms to transport toward the surface from the inner part of the ribbon matrix. Hence, the Zr1 ribbon has a higher η and k than Zr0 ribbon under the same condition. These results not only prove that Zr1 amorphous ribbon has high degradability to organic dyes, but also reveal that Zr element has the role of tunnel construction in Fe-based alloys.Based on the analysis of the elemental information, surface micro-morphology and micro-structure of Zr0 and Zr1 amorphous ribbons during MB solution (cZr, the as-spun Zr0, Zr0.5 and Zr1 amorphous ribbons have gradually increased degradation rate of MB solution. According to η and k than Zr0 and Zr0.5 ribbons under the same condition. The nonmonotonic influence of pH value and H2O2 concentration on the degradation η and k of Zr1 ribbon is similar to Zr0 and Zr0.5 ribbons. This work not only provides a new method for the remediation of organic dye wastewater, but also extends the application prospect of Fe-based amorphous alloys.In this work, we have prepared Zr0, Zr0.5 and Zr1 amorphous ribbons with melt-spun method and we studied the microstructure, MB solution degradation behavior with several technologies. With increasing