Data_Sheet_1_Improvement of thermostability and catalytic efficiency of xylanase from Myceliophthora thermophilar by N-terminal and C-terminal truncation.docx

IntroductionExtracting xylanase from thermophilic filamentous fungi is a feasible way to obtain xylanase with good thermal stability.MethodsThe transcriptomic data of Myceliophthora thermophilic destructive ATCC42464 were differentially expressed and enriched. By comparing the sequences of Mtxylan2 and more than 10 xylanases, the N-terminal and C-terminal of Mtxylan2 were truncated, and three mutants 28N, 28C and 28NC were constructed.Results and discussionGH11 xylan Mtxylan2 was identified by transcriptomic analysis, the specific enzyme activity of Mtxylan2 was 104.67 U/mg, and the optimal temperature was 65°C. Molecular modification of Mtxylan2 showed that the catalytic activity of the mutants was enhanced. Among them, the catalytic activity of 28C was increased by 9.3 times, the optimal temperature was increased by 5°C, and the residual enzyme activity remained above 80% after 30 min at 50–65°C, indicating that redundant C-terminal truncation can improve the thermal stability and catalytic performance of GH11 xylanase.</p

Structural Analysis of Low Molecular Weight Heparin by Ultraperformance Size Exclusion Chromatography/Time of Flight Mass Spectrometry and Capillary Zone Electrophoresis

Although low molecular weight heparins (LMWHs) have been used as anticoagulant agents for over 2 decades, their structures have not been fully characterized. In this work, we propose a new strategy for the comprehensive structural analysis of LMWHs based on the combination of ultraperformance size exclusion chromatography/electrospray quadruple time-of-flight-mass spectrometry (UPSEC/Q-TOF-MS) and capillary zone electrophoresis (CZE). More than 70 components, including oligosaccharides with special structures such as 1,6-anhydro rings, saturated uronic acid at the nonreducing end and odd-numbered saccharides units were identified with UPSEC/Q-TOF-MS. Furthermore, a more detailed compositional analysis was accomplished by CZE analysis. PEG10000 and MgCl₂ were added to the background electrolyte to separate those saccharides with the nearly same charge-to-mass ratio. Baseline separation and quantification of all the building blocks of the most complex LMWH, namely, enoxaparin, which include 10 disaccharides, 1 trisaccharide, 2 tetrasaccharides, and, of particular importance, 4 1,6-anhyro derivatives, was achieved using CZE for the first time. Additionally, the peaks of oligosaccharides, in the absence of commercially available standards, were assigned on the basis of the linear correlation between the electrophoretic mobilities of oligosaccharides and their charge-to-mass ratios. These two approaches are simple and robust for structural analysis of LMWHs

PEI-Mediated Assembly of Fe₃O₄ onto SiO₂‑Encapsulated CsPbBr₃ for Highly Sensitive Fluorescent Lateral Flow Immunoassay

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method’s sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL–1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL–1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL–1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation

Characterization of anti-TNF-alpha antibody.

<p>(A) Binding affinity of anti-mouse-TNF-alpha antibody. The equilibrium constant of anti-mTNF-alpha antibody 9C6 was measured by Surface Plasmon Resonance (SPR) using a CM5 sensor chip in a BIACORE3000 at 25°C. Recombinant mouse-TNF-alpha was captured in the flow cell. Running buffer and different concentrations of anti-mTNFa mAb 9C6 (0.64, 1.28, 2.56, 5.12, 10.24, and 20.48 µM diluted in running buffer) were applied to the antigen-containing flow cell for 3 min at a flow rate of 30 µl/min. The Kd of 9C6 was calculated as 1.4 nM. (B) Anti-mTNF-alpha mAb 9C6 neutralizes TNF-alpha-induced cytotoxicity in L929 cells. 3.5×10⁵ L929 cells were treated with 1 µg/ml actinomycin D and 3 ng/ml mouse TNF-alpha for 15 hours. Different dilutions of anti-mTNFa mAb 9C6 (with a starting concentration of 18 µg/ml) were added to the assay. Cell death was measured by the MTT assay (Thiazolyl Blue Tetrazolium Bromide staining, read at 570 nm) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006087#pone.0006087-Mosmann1" target="_blank">[20]</a>. (C) Anti-TNF-α mAb 9C6 protects mice from LPS-induced sepsis. 8 week-old C57BL/6 female mice, 8 mice/group, were injected intraperitoneally with 25 mg/kg LPS, along with 2.5 mg/kg anti-TNF-α mAb 9C6 or isotype control antibody. The number of surviving mice was recorded every six hours.</p

Generation of high affinity, biologically active anti-HMGB1 antibodies from NZB/W mice.

<p>(A) A T cell-specific tag fused to HMGB1 further increased immune response in NZB/W mice. NZB/W mice were immunized with GST-HMGB1 or HMGB1-MT. Serum titers were measured by ELISA using HMGB1-His-coated plates. Serum titer was defined as the highest dilution of serum at which the A450 ratio (A450 of sample/A450 of negative serum) was greater than 2.0. (B) Binding affinity and specificity of anti-HMGB1 antibodies.Antibody binding affinity was measured by ELISA using HMGB1-His-coated plates and the Kd of each antibody is indicated in the figure. Antibody specificity was measured by ELISA using the GST-HMGB1 Box (A), GST-HMGB1 Box (B) or GST-HMGB1 Box (A+B) as antigens. (C) Binding specificity of anti-HMGB-1 antibodies. Left panel: 0.3 µg of purified recombinant proteins (A+B:HMGB1-MT, A: GST-boxA, B:GST-boxB) were loaded and separated by electrophoresis on 10% SDS-PAGE gels. Western blots were carried out with 1% dry milk as the blocking buffer and 2 µg/ml purified antibodies as the primary antibody. The blots were then labeled with goat anti-mouse Ig-HRP and ECL substrate. 3 µg of purified recombinant proteins were separated by electrophoresis and stained with Coomassie Brilliant Blue for loading control. Right panel: 1×10⁷ Hela cells were lysed in 1 ml cell lysis buffer and mixed with 5× SDS sample buffer. 20 µl each of the above samples was separated by electrophoresis on 10% SDS-PAGE gels. Western blots were carried out with 5% BSA as the blocking buffer and 1 µg/ml biotinylated antibodies as the primary antibody. The blots were then labeled with streptavidin-HRP and ECL substrate. (D) anti-HMGB1 antibodies blocked HMGB1-induced IL-6 up-regulation. Raw264.7 cells were stimulated with 1 µg/ml HMGB1 for 8 hours, with or without 50 µg/ml of the antibodies indicated. Total mRNA was extracted with Trizol, and IL-6 mRNA levels were measured by real-time RT-PCR. Data are expressed as the means±SEM (n = 3). ** indicating p<0.01 when compared to samples treated with HMGB1 only. (E) Anti-HMGB1 mAb 3E8 protects mice from LPS-induced sepsis. 8 week-old C57BL/6 female mice, 8 mice/group, were injected intraperitoneally with 25 mg/kg LPS, along with three different doses of anti-HMGB1 antibody 3E8 or isotype control antibodies. The number of surviving mice was recorded every six hours.</p

Generation of high affinity, biologically active anti-MIF antibodies from NZB/W mice.

<p>(A) Use of NZB/W mice to break immune tolerance.NZB/W or BALB/C mice were immunized with His tagged MIF protein. Serum titers were measured by ELISA using GST-MIF coated plates. Serum titer was defined as the highest dilution of serum at which the A450 ratio (A450 of sample/A450 of negative serum) was greater than 2.0. (B) Competition assays between three anti-MIF clones, 10C3, 2A12 and 4E10. Human MIF was displayed on the surface of yeast cells. Yeast cells were incubated with 10 nM of biotin-labeled antibodies, with or without 100 nM of non-biotinylated antibodies as competitors. Streptavidin-PE was used for secondary labeling. (C) 10C3 inhibits MIF-induced nitric oxide secretion in a macrophage cell line. Raw264.7 cells were stimulated with recombinant MIF (3 µg/ml) and increasing concentrations of anti-MIF antibody 10C3 for 16 hours. Nitric oxide concentration in the supernatant was detected using the Griess assay. (D) 10C3 protects mice from LPS-induced sepsis. 8 week C57BL/6 female mice, 10 mice/group, were injected intraperitoneally with 22.5 mg/kg LPS, along with anti-MIF antibody 10C3 or isotype control antibodies (5 mg/kg). The number of surviving mice was recorded every six hours.</p

DataSheet1_The characteristic and size–frequency distribution of rocks at the Zhurong landing site, Mars.pdf

The rock characteristic and size–frequency distribution (SFD) on Mars are important for understanding the geologic and geomorphic history of the surface, for evaluating the trafficability of roving, and for planning the potential infrastructure construction. Tianwen-1, China’s first autonomous Mars exploration mission, formed an excavated depression during touchdown, which has been the deepest depression on the Martian surface so far compared with others. According to the images captured using the Navigation and Terrain Cameras (NaTeCams) onboard the rover, Zhurong, the SFD of rocks is calculated and compared inside the excavated depression, within and out of the blast zone. For the first time, the rock size distribution inside the excavated depression is obtained, exposing the geological features of the shallow subsurface on Mars at a depth of tens of centimeters, which will surely be important for future drilling missions. It is found that the rock abundance in the depression is smaller than the original abundance on the surface, and the distribution of rocks in the blast zone on the surface is greatly influenced by the touchdown. In addition, based on the fractal dimension of rock sizes, the rocks (>10 mm) at the shallow subsurface of the Zhurong landing site may experience two different geological processes.</p

Additional file 1 of USP40 promotes hepatocellular carcinoma cell proliferation, migration and stemness by deubiquitinating and stabilizing Claudin1

Additional file 1. Supplementary Tables