Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis–Menten constants <i>K</i>_m for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine <i>K</i>_m and turnover number <i>k</i>_cat for trypsin digestion of angiotensin II (<i>K</i>_m and <i>k</i>_cat are determined to be 6.4 mM and 1.3 s^–1, respectively)

Mass-Based Photothermal Comparison Among Gold Nanocrystals, PbS Nanocrystals, Organic Dyes, and Carbon Black

Gold nanocrystals have attractive plasmon-enabled photothermal conversion properties, which have been widely employed for photothermal therapy and solar energy harvesting. For practical applications, the mass-normalized photothermal conversion performance is often desired to be known for Au nanocrystals with different shapes and sizes and for different nanomaterials. We study the photothermal conversion performances of differently shaped and sized Au nanocrystals and compare them with those of PbS nanocrystals, carbon black, and organic dyes at the same mass concentrations. Both the mass-normalized extinction cross section and the photothermal conversion efficiency of Au nanocrystals decrease as their size is increased. The photothermal conversion performance of carbon black is comparable to that of relatively small Au nanocrystals, while the photothermal conversion performance of organic dyes and PbS nanocrystals is inferior to that of Au nanocrystals. Our results are useful for the design of Au nanocrystals and the choice of nanomaterials for photothermal applications

Data_Sheet_1_Transcriptomic and metabolomic analyses to study the key role by which Ralstonia insidiosa induces Listeria monocytogenes to form suspended aggregates.zip

Ralstonia insidiosa can survive in a wide range of aqueous environments, including food processing areas, and is harmful to humans. It can induce Listeria monocytogenes to form suspended aggregates, resulting from the co-aggregation of two bacteria, which allows for more persistent survival and increases the risk of L. monocytogenes contamination. In our study, different groups of aggregates were analyzed and compared using Illumina RNA sequencing technology. These included R. insidiosa under normal and barren nutrient conditions and in the presence or absence of L. monocytogenes as a way to screen for differentially expressed genes (DEGs) in the process of aggregate formation. In addition, sterile supernatants of R. insidiosa were analyzed under different nutrient conditions using metabolomics to investigate the effect of nutrient-poor conditions on metabolite production by R. insidiosa. We also undertook a combined analysis of transcriptome and metabolome data to further investigate the induction effect of R. insidiosa on L. monocytogenes in a barren environment. The results of the functional annotation analysis on the surface of DEGs and qPCR showed that under nutrient-poor conditions, the acdx, puuE, and acs genes of R. insidiosa were significantly upregulated in biosynthetic processes such as carbon metabolism, metabolic pathways, and biosynthesis of secondary metabolites, with Log2FC reaching 4.39, 3.96, and 3.95 respectively. In contrast, the Log2FC of cydA, cyoB, and rpsJ in oxidative phosphorylation and ribosomal pathways reached 3.74, 3.87, and 4.25, respectively. Thirty-one key components were identified while screening for differential metabolites, which mainly included amino acids and their metabolites, enriched to the pathways of biosynthesis of amino acids, phenylalanine metabolism, and methionine metabolism. Of these, aminomalonic acid and Proximicin B were the special components of R. insidiosa that were metabolized under nutrient-poor conditions.</p

Polydiacetylene-Polymethylmethacrylate/Graphene Composites as One-Shot, Visually Observable, and Semiquantative Electrical Current Sensing Materials

Aiming to develop pH-paper-like current sensing materials, we prepared irreversible electrochromic PDA-PMMA/graphene composites. The composites exhibited an excellent linear relationship between critical responsive currents and the amount of graphene in the system. In these composites, PDA acted as the electrochromic material and graphene as the conductive matrix. The presence of PMMA not only ensured mechanical performance but also made the color change more obvious to be observed by the naked eye

Additional file 1: FigureS1 of Down-regulation of S100A9 inhibits osteosarcoma cell growth through inactivating MAPK and NF-ÎşB signaling pathways

The knockdown of S100A9 had no effect on the cell apoptosis, RAGE or TLR4. A. The apoptosis was tested after knockdown of S100A9 (n=3). A histogram about the sum of the upper right quadrant(Q2) and the lower right quadrant(Q3) was shown in Supplement fig.1A. Q2 represents late apoptotic cells, Q3 represents early apoptotic cells. B. The protein levels of RAGE and TLR4 were tested by western blot in three OS cell lines (group1-blank control, group2-transfected with empty vectors, group3-transfected with siRNA-S100A9 vectors; n=3). (TIFF 5193 kb

Additional file 2: FigureS2 of Down-regulation of S100A9 inhibits osteosarcoma cell growth through inactivating MAPK and NF-κB signaling pathways

The up-regulation of S100A9 enhanced osteosarcoma proliferation, migration and invasion.A.The protein levels of S100A9 were tested by western blot in the three OS cell lines (group1-blank control, group2-transfected with empty vectors, group3-transfected with S100A9 vectors; n=3, ***P < 0.001,versus empty vectors group). B. The mRNA levels of S100A9 were tested by real-time Quantitative PCR in the three OS cell lines (n=9, ***P < 0.001, versus empty vectors group). C. CCK-8 was used to assess the three OS cell lines in 1, 2, 3 and 4 days (n=15, *p < 0.05, **P < 0.01; ***P <  0.001, versus empty vectors group at the same time points). D. Cell cycle distribution was tested in the group1, 2 and 3 using flow cytometry; the histogram of cell cycle distribution was shown(n=3, **P < 0.01, versus empty vectors group at G0/G1 phase; ##P < 0.01, versus empty vectors group at S phase). E. The histogram of cell migration assay were evaluated by transwell chambers in group1, 2 and 3(n=5,***P < 0.001, versus empty vectors group). F. The histogram ofcell invasion assay were evaluated by transwell chambers in group1, 2 and 3 (n=5, ***P < 0.001, versus empty vectors group).(TIFF 10616 kb

Receptor-Free Poly(phenylenevinylene) Fibrous Membranes for Cation Sensing: High Sensitivity and Good Selectivity Achieved by Choosing the Appropriate Polymer Matrix

Poly­(phenylenevinylene)/polyimide (PPV/PI) and poly­(phenylenevinylene)/ polymethylmethacrylate (PPV/PMMA) fibrous membranes without any deliberately introduced receptors were prepared as fluorescence sensing materials through electrospinning, followed by thermal treatment. Both of these membranes displayed higher sensitivity toward most cations compared to the corresponding spin-coated films. PPV/PMMA membranes were more sensitive than PPV/PI membranes toward Cu²⁺ and Fe³⁺. About 4.5 fold of intensity enhancement upon 20 nM of Cu²⁺, 80% of quenching upon 20 nM of Fe³⁺ with fast response and simple regeneration were realized for PPV/PMMA membrane. The preliminary investigation into the mechanism revealed that the properties of the polymer matrix and thermal treatment of the membrane played important roles in the sensing performance

A Unique Chemo-photodynamic Antitumor Approach to Suppress Hypoxia via Ultrathin Graphitic Carbon Nitride Nanosheets Supported a Platinum(IV) Prodrug

Tumor hypoxia severely restrains the efficiency of irreversible O2-consumption photodynamic therapy. The deep hypoxia induced by photodynamic therapy can promote the level of hypoxia inducible factor 1α that participates in many tumor processes and eventually lead to poor therapeutic outcomes. Herein, a chemo-photodynamic antitumor strategy based on ultrathin graphitic carbon nitride nanosheets loaded with a hypoxia-targeting platinum(IV) prodrug is reported. Under low-intensity visible light irradiation, such integrated nanosheets effectively generate reactive oxygen species together with DNA binding platinum species to achieve enhanced antiproliferation efficacy by downregulating HIF-1α under hypoxic conditions

DataSheet_1_Causal relationship between the blood immune cells and intervertebral disc degeneration: univariable, bidirectional and multivariable Mendelian randomization.zip

BackgroundIntervertebral disc degeneration (IVDD) is a prominent contributor to chronic low back pain, impacting millions of individuals annually. Current research on disc degeneration is placing a growing emphasis on the role of the immune system in this process. Nevertheless, the precise relationship between immunity and disc degeneration remains to be fully elucidated.MethodWe obtained GWAS data for immune cells from the latest summary-level GWAS, including 6,620 individuals from Sardinian and 746,667 individuals from five global populations. Summary results for IVDD were sourced from the FinnGen consortium, comprising 20,001 cases and 164,682 controls. We conducted a comprehensive univariable Mendelian randomization (MR) analysis to explore the potential causal relationship between immune cells and IVDD. Primary estimation was carried out using Inverse-Variance Weighting (IVW). To ensure robustness, we employed additional MR methods such as MR-Egger, Weighted Median, Weighted Mode, and Simple Mode. Various tests were employed to assess pleiotropy and heterogeneity, including the Cochran Q test, leave-one-out test, MR-Egger intercept analysis and MR-PRESSO test. To account for potential confounding factors among the immune cells, we conducted a multivariable MR analysis. Finally, we investigated the possibility of a reverse association between immune cells and IVDD through bidirectional MR.ResultIn total, our study identified 15 immune cells significantly associated with IVDD through univariable MR. Among these, 9 immune cell types were indicated as potential contributors to IVDD, while 6 were found to have protective effects. Importantly, we observed no evidence of heterogeneity or pleiotropy, signifying the robustness of our results. To mitigate confounding among immune cells, we utilized multivariable MR, leading to the discovery that only 9 immune cell types exerted independent effects on IVDD. These encompassed 7 as risk factors and 2 as protective factors. Additionally, our analysis revealed a bidirectional causal relationship between CD39+ CD4+ T cell �4+ T cell and IVDD.ConclusionOur findings suggest a connection between immune cells and the risk of IVDD, shedding light on potential therapeutic avenues for modulating immune cell function in individuals with IVDD. However, the specific underlying mechanisms warrant further investigation in future experiments.</p

Up-Regulation of microRNA-126 May Contribute to Pathogenesis of Ulcerative Colitis via Regulating NF-kappaB Inhibitor IκBα

<div><h3>Background</h3><p>MicroRNAs (miRNAs) are important post-transcriptional regulators. Altered expression of miRNAs has recently demonstrated association with human ulcerative colitis (UC). In this study, we attempted to elucidate the roles of miR-126 in the pathogenesis of UC.</p> <h3>Methods</h3><p>Expression of miR-126, miR-21, miR-375 and the potential targets NF-κB inhibitor alpha (IκBα, IKBA or NFKBIA), Polo-like kinase 2 (PLK2) and v-Crk sarcoma virus CT10 oncogene homolog (CRK) were assessed in 52 colonic biopsies from patients with active UC, inactive UC, irritable bowel syndrome (IBS) and from healthy subjects by quantitative RT-PCR and immunofluorescence analyses. Regulation of gene expression by miR-126 was assessed using luciferase reporter construct assays and specific miRNA mimic transfection.</p> <h3>Results</h3><p>We found that the expression of miR-126 and miR-21 were significantly increased in active UC group compared to the inactive UC, IBS and healthy control groups (<em>P</em><0.05). In contrast, the expression of IKBA mRNA and protein was remarkably decreased in the active UC group compared with the other three groups (<em>P</em><0.05). The expression of miR-126 and IKBA mRNA were inversely correlated in active UC patients (<em>P</em><0.05). However the expression of miR-375, PLK2 and CRK showed no difference between each group. Furthermore, we demonstrate that endogenous miR-126 and exogenous miR-126 mimic can inhibit IκBα expression. Finally, mutating the miR-126 binding site of the IKBA 3′-UTR reporter construct restored reporter gene expression.</p> <h3>Conclusion</h3><p>miR-126 may play roles in UC inflammatory activity by down-regulating the expression of IKBA, an important inhibitor of NF-κB signaling pathway.</p> </div