Journal: Cell

Article Title: Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted strategy for bacterial vaginosis treatment

doi: 10.1016/j.cell.2024.07.029

Figure Lengend Snippet: Detection of enzymatic products from ohyA orthologs and characterization of ohyA9 and farE genetic knockouts in L. gasseri , related to Figures 4 and (A) MS2 spectra with major fragmentation labels for 10-HSA standard (Ambeed, A125712-50MG). (B) Extracted ion chromatograms from supernatants of ΔSaohyA /empty vector, ΔSaohyA /p SaohyA , ΔSaohyA /pLCRIS_00558, and ΔSaohyA /pLCRIS_00661 cultured with LOA for 1 h. Annotated peaks include LOA (18:2) and the detected hydroxyFA ( h 18:1). (C) OhyA9 enzymatic activity reaction diagram with LOA substrate. (D) MS2 spectra with major fragmentation labels for the h18:1 peak from ΔSaohyA /pLCRIS_00661 cultured with LOA (lower right of Figure S4 B), identified as 10-hydroxy-12-octadecenoic acid ( h 18:1). (E) OhyA12 enzymatic activity reaction diagram with LOA substrate. (F) MS2 spectra with major fragmentation labels for the h18:1 peak from ΔSaohyA /pLCRIS_00558 cultured with LOA (lower left of Figure S4 B), identified as 13-hydroxy-9-octadecenoic acid. (G) 13 C 18 -10-HSA concentrations in cell pellets of L. crispatus , L. gasseri , and L. jensenii cultured for 72 h in NYCIII broth with and without 13 C 18 -OA (3.2 mM). The cell pellets are from the same cultures as the supernatants shown in Figure 3 E. (H) 13 C 18 -10-HSA concentrations in cell pellets of L. crispatus and L. iners cultured for 72 h in NYCIII broth with and without 13 C 18 -OA (100 μM, which is a sublethal concentration for L. iners ). The cell pellets are from the same cultures as the supernatants shown in Figure 3 F. (I) Presence of gene functions predicted to encode oleate hydratase ( ohyA ) and putative fatty acid efflux pump ( farE ) activity in long-read sequenced, isolate genomes of the indicated FGT species. Presence of gene functions involved in exogenous fatty acid acquisition and utilization ( fakAB , plsC , plsX , and plsY ) is shown for comparison. (J) Untargeted lipidomics was performed on control (blank) media and spent media supernatants collected from strains of diverse FGT bacteria after 72 h of culture in NYCIII broth. Blank media supplemented with 100 μM OA is shown as a positive control. Changes in concentration of key LCFA metabolites for each bacterial species are shown as the log 10 (fold change) of their median relative abundances compared with control media. The plot depicts median fold change values for 5 technical replicates per condition. Statistical significance was determined by unpaired t test using the Bonferroni method to correct for multiple hypothesis testing ( ∗ adjusted p < 0.05). (K) Representative h FA extracted ion chromatograms for human CVL samples with CT1 ( L. crispatus -dominant, top) or CT2 ( L. iners -dominant, bottom) bacterial communities, quantified via a targeted metabolomics approach employing picolylamine-based derivatization. , (L) DNA gel of PCR products amplified from the primers flanking either LGAS_1630 ( farE ) or LGAS_1351 ( ohyA9 ) from the L. gasseri ATCC 33323 wild-type (WT) strain and ΔohyA9 and ΔfarE genetic knockout (KO) strains. Expected amplicon lengths were 3.9 kb for LGAS_1630 in WT, 2.0 kb for LGAS_1351 in WT, while in-frame gene deletions of LGAS_1351 in ΔohyA9 and of LGAS_1630 in ΔfarE each had expected amplicon lengths of 1.2 kb. KO strains were additionally WGS verified. (M) Cultures of L. gasseri WT, L. gasseri Δ farE , and L. gasseri Δ farE /p farE were grown to mid- to late-log phase and exposed to varying concentrations of OA, then ATP release assays were performed. (N) Detection of 13 C-labeled OA ( 13 C 18 -OA) in pellets of L. gasseri Δ farE and Δ farE /p farE genetic mutant strains treated with 100 and 400 μM 13 C 18 -OA in NYCIII broth for 15 min. Pellets were washed two times with ice-cold PBS before sample preparation for targeted 13 C 18 -OA detection. The fold change of [ 13 C 18 -OA] was calculated by dividing the relative abundance of 13 C 18 -OA detected in the 400 μM 13 C 18 -OA condition for each technical replicate by the median relative abundance of 13 C 18 -OA detected in 100 μM 13 C 18 -OA condition. Significance of the difference in fold change was determined by unpaired t test ( ∗∗∗ p < 0.001). Points represent 5 technical replicates per condition. (O) 13 C 18 -10-HSA relative abundance in cell pellets from L. gasseri WT, ΔohyA9 , ΔohyA9 /p ohyA9 , ΔfarE , and ΔfarE /p farE cultured for 24 h in NYCIII broth with and without 13 C 18 -OA (100 μM). Pellets are from the same cultures as the supernatants in Figure 5 D. Points represent 2 or 3 technical replicates per condition. (G and H) Points represent 3 technical replicates per condition.

Article Snippet: Statistical significance was determined by unpaired t test using the Bonferroni method to correct for multiple hypothesis testing ( ∗ adjusted p < 0.05). (K) Representative h FA extracted ion chromatograms for human CVL samples with CT1 ( L. crispatus -dominant, top) or CT2 ( L. iners -dominant, bottom) bacterial communities, quantified via a targeted metabolomics approach employing picolylamine-based derivatization., (L) DNA gel of PCR products amplified from the primers flanking either LGAS_1630 ( farE ) or LGAS_1351 ( ohyA9 ) from the L. gasseri ATCC 33323 wild-type (WT) strain and ΔohyA9 and ΔfarE genetic knockout (KO) strains.

Techniques: Plasmid Preparation, Cell Culture, Activity Assay, Concentration Assay, Comparison, Control, Bacteria, Positive Control, Amplification, Knock-Out, Labeling, Mutagenesis, Sample Prep

Journal: Dentomaxillofacial Radiology

Article Title: Assessment of cone-beam CT technical image quality indicators and radiation dose for optimal STL model used in visual surgical planning

doi: 10.1093/dmfr/twae026

Figure Lengend Snippet: Classification of the CBCT and CT protocols based on the CNR and MTF values.

Article Snippet: Furthermore, CT images were acquired using 2 CT scanners; CT1: GE BrightSpeed 16-slice (GE Healthcare Technologies, Waukesha, WI, United States) and CT2: GE Revolution 256-slice (GE Healthcare Technologies, Waukesha, WI, United States).

Techniques:

Journal: Dentomaxillofacial Radiology

Article Title: Assessment of cone-beam CT technical image quality indicators and radiation dose for optimal STL model used in visual surgical planning

doi: 10.1093/dmfr/twae026

Figure Lengend Snippet: The exposure parameters, CNR, MTF (10%), effective dose, bone volume, and area of CBCT and MSCT scanners.

Article Snippet: Furthermore, CT images were acquired using 2 CT scanners; CT1: GE BrightSpeed 16-slice (GE Healthcare Technologies, Waukesha, WI, United States) and CT2: GE Revolution 256-slice (GE Healthcare Technologies, Waukesha, WI, United States).

Techniques: