lc nexera 40 series uhplc hplc system  (Shimadzu Corporation)

Journal: Synthetic and Systems Biotechnology

Article Title: Functional characterization of four glycosyltransferases for biosynthesis of steroidal saponins in medicinal plant Paris polyphylla

doi: 10.1016/j.synbio.2026.04.002

Figure Lengend Snippet: Reaction schemes and LC-MS analysis of Pp UGTs reactions with diosgenin and pennogenin . ( a ) Biosynthesis of trillin mediated by UGT91BP2 and UGT703R1–3 through transferring glucose from UDP-glucose to the C-3 OH position of diosgenin. ( b ) Extracted ion chromatograms (EICs) of the in vitro enzyme activity assays of recombinant UGT91BP2 and UGT703R1–3 with UDP-glucose and diosgenin showing the formation of trillin, as compared to the authentic trillin standard. Control is the empty expression vector. ( c ) MS spectra of the enzymatic reaction products in hydrogen and sodium ion adducts compared to the fragmentation pattern of the trillin standard. The product molecular ions [M + H] + and [M +Na] + and the feature fragment ion [M + H − Glc] + with m / z 415.32 are marked. Of note, additional peaks observed in the diosgenin EIC ( m / z 415.32) for UGT703R1–3 might correspond to substrate isomeric impurities, in-source adducts, or minor non-enzymatic by-products; these peaks, which arose from the extracted ion flow of the substrate m / z , did not co-elute with the product, trillin , and therefore did not affect the interpretation of the product formation. ( d ) Biosynthesis of pennogenin 3- O -glucoside mediated by the UGT703R1–3 through transferring glucose from UDP-glucose to the C-3 OH position of pennogenin. ( e ) EICs of the in vitro enzyme activity assays of recombinant UGT91BP2 and UGT703R1–3 with UDP-glucose and pennogenin showing the formation of pennogenin 3- O -glucoside, compared to the control empty expression vector. ( f ) MS/MS spectra ([M + H – Glc] + and [M + H – Glc − H 2 O] + ) of the enzymatic reaction products.

Article Snippet: Liquid chromatography-mass spectrometry (LC-MS) was equipped with a YMC-Triart C18 column (250 mm × 4.6 mm, 5 μm), and the column temperature was set to 30 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Transferring, In Vitro, Activity Assay, Recombinant, Control, Expressing, Plasmid Preparation, Tandem Mass Spectroscopy

Journal: Biofilm

Article Title: Amino acid starvation and iron limitation facilitate the biofilm formation of Klebsiella pneumoniae within urine

doi: 10.1016/j.bioflm.2026.100347

Figure Lengend Snippet: The roles of amino acid starvation regulating c-di-GMP signaling and biofilm formation during the growth of K. pneumoniae in urine. (a) RT-qPCR was performed to assess the expression levels of 10 c-di-GMP-associated genes, comprising 7 upregulated and 3 downregulated genes, as identified through RNA-Seq analysis. Each experiment was conducted in triplicate, with data normalized against the housekeeping gene gapA . Relative expression changes were determined using the 2 −ΔΔCT method. (b) Measurement of intracellular c-di-GMP levels by LC-MS/MS in K. pneumoniae cultured in the indicated media. Error bars indicate the mean ± SD from three biologically independent samples. (c) Biofilm formation quantified by OD 595 measurements after crystal violet staining of K. pneumoniae cultured in the indicated media. Data represent means ± SD of triplicate samples. (d) Images of biofilms formed by K. pneumoniae under different culture conditions. The results were analyzed as described in e. (e) Proposed model of c-di-GMP regulation of K. pneumoniae biofilm formation in response to amino acid starvation during growth in urine. Biofilm control is depicted as a multilateral coordination module within the c-di-GMP response network referred to the latest research reports [ , , , , , , , ]. Blue arrows denote activation, while red T-bar lines denote inhibition. (f) The upregulation of cellulose, type III pili (T3P), and K-capsule in K. pneumoniae under amino acid starvation in urine. RT-qPCR data were analyzed as described in a. Statistical significance was assessed using two-way ANOVA, comparing mutant strains to the wild-type strain cultured in LB medium (∗∗ P < 0.01, ∗∗∗ P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Quantification was performed on an AB SCIEX QTRAP 6500+ LC-MS/MS instrument [ ], and measured nucleotide levels were expressed relative to the bacterial counts of the corresponding samples to provide normalized intracellular c-di-GMP values.

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Cell Culture, Staining, Control, Activation Assay, Inhibition, Mutagenesis