trimethoprim  (Gold Biotechnology Inc)

Journal: bioRxiv

Article Title: Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

doi: 10.1101/2024.06.12.598711

Figure Lengend Snippet: (a) Maximum intensity projection image of MMC-induced mNeonGreen Phollow virocells before (top) and after a lysis event (bottom). Green arrowheads indicate a cell that gives rise to a membrane vesicle containing virion aggregates and cytosolic SpyCatcher protein. (b) Top: MMC-induced cultures of E. coli carrying “dark” Phollow phages. Staining the culture with the membrane dye FM 4-64 and DNA dye Hoechst 33342 reveals that numerous membrane vesicles are generated during cell lysis, which frequently contain DNA (green arrowheads). Bottom: Purified lysates show dramatically reduced levels of membrane vesicles and regularly sized DNA-positive puncta that are likely individual virions. (c) Representative flow virometry plots showing the gating strategy for quantifying Phollow phage virions in purified lysates. Left: A MMC-treated E. coli HS population cured of their P2 prophage and expressing an mNeonGreen (mNG) catcher peptide establishes a baseline for fluorescent viral-like particles. Middle: A MMC-treated E. coli HS population carrying a wild-type P2 prophage and expressing an mNG catcher peptide is used to establish a generic “Debris” gate resulting from phage-driven cellular lysis. Right: A MMC-treated E. coli HS mNG Phollow virocell population is used to quantify production of fluorescently marked viral-like particles. (d) Representative flow virometry plots showing gates for AausFP1 (left) and mKate2 (right) Phollow phage virions. Gates were set as in panel c but “Debris” gates are not shown. (e) Representative flow virometry plot showing the quantification of Phollow phage virions from a mixed lysate. (f) Fluorescence microscopy-based quantification of Phollow phage virion production in response to treatment with the genotoxic antibiotics mitomycin C (left), ciprofloxacin (middle), or trimethoprim (right). Antibiotic concentrations are given relative to each antibiotic’s minimum inhibitory concentration (MIC) against wild-type E. coli HS. Each circle represents a distinct and non-overlapping field of view; data were pooled from 3 biological replicates. Data for the “0” concentration is the same for all plots and is used to set a statistical baseline. Statistical groupings (denoted by letters and color-coding) in each plot were determined using one-way ANOVA with a Kruskal-Wallis and Dunn’s multiple comparisons test (p < 0.05). (g) Flow virometry-based quantification of Phollow phage virion production induced by mitomycin C (MMC, 0.1x MIC), ciprofloxacin (Cip, 0.5x MIC), or trimethoprim (Tri, 0.5x MIC). Bars indicate medians derived from three biological replicates (circles). No statistical differences were found by one-way ANOVA using a Kruskal-Wallis and Dunn’s multiple comparisons test (p < 0.05).

Article Snippet: To evaluate the minimum inhibitory concentration (MIC) of MMC, trimethoprim (GoldBio), and ciprofloxacin (GoldBio), Phollow virocells were first grown overnight in 5 mL TSB at 37° C with shaking.

Techniques: Lysis, Membrane, Staining, Generated, Purification, Expressing, Fluorescence, Microscopy, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

doi: 10.1101/2024.06.12.598711

Figure Lengend Snippet: (a-c) Optical density at 600 nm measurements of E. coli HS cultures in response to various concentrations of (a) MMC, (b) ciprofloxacin, and (c) trimethoprim. Data represent the average of three independent endpoint measurements after 24 hours of incubation at 37°C, plotted as heatmaps. Arrowheads in each panel indicate the minimum inhibitory concentration (MIC), defined as the minimum concentration required to significantly impair bacterial growth without complete eradication.

Article Snippet: To evaluate the minimum inhibitory concentration (MIC) of MMC, trimethoprim (GoldBio), and ciprofloxacin (GoldBio), Phollow virocells were first grown overnight in 5 mL TSB at 37° C with shaking.

Techniques: Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

doi: 10.1101/2024.06.12.598711

Figure Lengend Snippet: (a) Top: Schematic of a larval zebrafish with relevant anatomical features highlighted. Bottom: Maximum intensity projection of a 7-day old larval zebrafish colonized by E. coli generated from 297 tile-scanned and merged images acquired by super-resolution microscopy. (right) Inset is a single image of an intestinal region containing luminal aggregates of E. coli (arrowhead). DNA (magenta, JF549 dye) and actin (purple, CF405-phalloidin) highlight intestinal structure. (b) Diagram of bacterial colonization and antibiotic induction timeline. “Tri” = Trimethoprim. (c) (i) Maximum intensity projection image of an untreated E. coli Phollow virocell population within the gut (white arrowhead). (ii, iii) Two example maximum intensity projection images of trimethoprim-treated E. coli Phollow virocell populations. White arrowheads mark bacterial aggregates and single cells; black arrowheads mark viral particles. Images were acquired 4h post-treatment. (d) Maximum intensity projection image showing viral particles in the esophageal region. White arrowhead marks a single cell; black arrowhead marks viral particles. Image was acquired 4h post-treatment. (e) Fluorescence microscopy images of water samples taken from untreated (left) or trimethoprim-treated (right) zebrafish. An abundance of viral particles is evident in treated samples. Images were acquired 4h post-treatment. (f) Quantification of infectious virions from dissected intestinal tissues (left) and water (right) at 4h and 24h post-trimethoprim treatment (cyan circles). Quantification of infectious virions from time-matched untreated samples are shown in gray. Infectious virions are estimated by measuring the number of lysogen-forming units (LFU) per gut or ml of water. Statistical groupings (denoted by letters) in each plot were determined using one-way ANOVA with a Kruskal-Wallis and Dunn’s multiple comparisons test (p < 0.05). (g) Maximum intensity projection image showing viral particles derived from Plesiomonas Phollow virocells within the intestine. White arrowhead marks a single cell; black arrowhead marks viral particles. Image was acquired 4h post-treatment. (h) Maximum intensity projection image showing viral particles derived from Plesiomonas Phollow virocells within enteroendocrine cells (EEC, green). The membranes of EECs are marked using TgBAC( nkx2.2a : megfp ) reporter fish. Black arrowheads mark EECs containing viral particles or phage debris. Inset shows a cartoon representation of viral particles associating with an EEC. Image was acquired 4h post-treatment. (i) Left: Maximum intensity projection image showing viral particles derived from Plesiomonas Phollow virocells associating with hepatocytes of the liver. Hepatocytes can be disguised based on the circular morphology of their nuclei . White arrowhead marks virions within a vesicle-like structure; black arrowhead marks a virion aggregate or single phage. Inset shows a cartoon representation of viral particles associating with hepatocytes. Right: montage shows liver tissue from a separate fish stained with BDP (green) to mark lipid droplets. Image was acquired 4h post-treatment. (j) Maximum intensity projection image showing viral particles derived from Plesiomonas Phollow virocells within a blood vessel in the brain. White arrowhead marks a nucleated red blood cell; black arrowhead marks virions or phage debris associating with the surface of blood cells. Note: the opacity of brain tissue made it difficult to determine the presence of individual viral particles in this sample. Inset shows a cartoon representation of viral particles associating with red blood cells. Image was acquired 4h post-treatment.

Article Snippet: To evaluate the minimum inhibitory concentration (MIC) of MMC, trimethoprim (GoldBio), and ciprofloxacin (GoldBio), Phollow virocells were first grown overnight in 5 mL TSB at 37° C with shaking.

Techniques: Generated, Super-Resolution Microscopy, Fluorescence, Microscopy, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

doi: 10.1101/2024.06.12.598711

Figure Lengend Snippet: (a) Maximum intensity projection image showing viral particles derived from untreated E. coli HS Phollow virocells undergoing spontaneous phage lytic replication within the intestine. White arrowhead marks bacterial cells; black arrowhead marks viral particles. (b) Representative fluorescent microscopy images of AausFP1 E. coli HS Phollow virocell in zebrafish embryo media (EM) in the absence of fish. Bacteria were separated from fish immediately before the experiment to allow for the diffusion of fish-derived nutrients and other factors. Subsequently, bacteria were treated with trimethoprim and incubated at 28.5°C for 4h. Bacterial cells under these conditions do not display viral foci or signs of phage lytic replication (white arrowhead). (c) Maximum intensity projection images of MMC-induced Plesiomonas Phollow virocells. Each panel shows a representative image of fluorescent viral foci production and organization, which are similar to those in E. coli Phollow virocells. Magenta dashed lines mark the cell perimeter.

Article Snippet: To evaluate the minimum inhibitory concentration (MIC) of MMC, trimethoprim (GoldBio), and ciprofloxacin (GoldBio), Phollow virocells were first grown overnight in 5 mL TSB at 37° C with shaking.

Techniques: Derivative Assay, Microscopy, Bacteria, Diffusion-based Assay, Incubation

Journal: bioRxiv

Article Title: Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals

doi: 10.1101/2024.06.12.598711

Figure Lengend Snippet: (a) Diagram of bacterial colonization and antibiotic induction timeline. “Tri” = Trimethoprim. (b) Enumeration of community composition by differential plating. Data are presented as average relative abundances; bars indicate standard error of the mean (SEM) from across 3 biological replicates at each time point and condition. (c) Fluorescent microscopy images of AausFP1 virocell/mKate2 target cell gut communities prior to (top), 2h post- (middle), and 4h post-trimethoprim treatment. Each image was taken from different zebrafish hosts. Black arrowheads in the middle image indicate areas containing lytically replicating phage. Inset in the bottom panel shows the AausFP1 virocell and mKate2 target cell channels separately to highlight degree of community mixing. (d) Representative fluorescence microscopy image of a water sample at 4h post-trimethoprim treatment. White arrowhead indicates a virion aggregate; Black arrowhead indicates a single viral particle. (e) Fluorescent microscopy images of AausFP1 virocell/mKate2 target cell gut communities prior to (top) and 2h after (bottom) a 2 nd post-trimethoprim treatment. Each image is from different zebrafish hosts. (f) Representative fluorescence microscopy image of a water sample 2h after a 2 nd trimethoprim treatment. Magenta arrowhead indicates an mKate2 Phollow phage virion; green arrowhead indicates an AausFP1 Phollow phage virion.

Article Snippet: To evaluate the minimum inhibitory concentration (MIC) of MMC, trimethoprim (GoldBio), and ciprofloxacin (GoldBio), Phollow virocells were first grown overnight in 5 mL TSB at 37° C with shaking.

Techniques: Microscopy, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A high-light therapy restores the circadian clock and corrects the pathological syndrome generated in restricted-fed mice

doi: 10.1073/pnas.2403770121

Figure Lengend Snippet: A “high-light” treatment shifts the SCNCC and locomotor activity by 12 h in time RF mice. ( A ) Schematic representation showing the administration of a T 0-12 150–T 12-0 700 “high-light” regime to RF mice. ( B ) Relative RNA transcript levels (Q-RT-PCR) of CC components in SCN tissue. Mice subjected to food ad libitum and animal facility light condition (T 0-12 150–T 12-0 0) are represented in black, while mice subjected to RF (food available from ZT0–ZT12) for 5 wk under a T 0-12 150–T 12-0 0 light condition are represented in orange, and mice subjected to 4 wk of RF followed by 1 wk of RF under a T 0-12 150–T 12-0 700 “high-light” regime are represented in blue. ( C ) Actimetric analyses of the circadian locomotor activity of mice treated as under ( B ). ( D and E ) Relative transcript levels of CC components in liver ( D ) and ileum ( E ) of mice treated as in ( B ). All values are mean ± SEM.

Article Snippet: Experiments were performed on 8- to 12-wk-old C57BL6/J wild-type male mice (Charles River Laboratories) under light–dark condition (T ZT0-ZT12 150–T ZT0-ZT12 0 lx) and light-high conditions (i.e., T ZT0-ZT12 150–T ZT0-ZT12 700 lx, T ZT0-ZT12 75–T ZT0-ZT12 150 lx, and T ZT0-ZT12 350–T ZT0-ZT12 700 lx).

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction