Journal: Molecules

Article Title: Phages and Enzybiotics in Food Biopreservation

doi: 10.3390/molecules26175138

Figure Lengend Snippet: Phages tested against food-borne pathogens and their proposed use as food biopreservatives.

Article Snippet: Listeria monocytogenes , A500 ATCC ® 23074-B1TM , L. monocytogenes isolated from Guinea pig , TEM , 38,867 bp , Siphoviridae , Control of L-forms of L. monocytogenes on surfaces , [ , ] .

Techniques: Bacteria, Sequencing, Inhibition, Control, DNA Sequencing, Infant Formula, Pulsed-Field Gel, Isolation, Environmental Sampling, Amplification

Journal: Molecules

Article Title: Phages and Enzybiotics in Food Biopreservation

doi: 10.3390/molecules26175138

Figure Lengend Snippet: Enzybiotics tested against food-borne pathogens and their proposed use in foods.

Article Snippet: Listeria monocytogenes , A500 ATCC ® 23074-B1TM , L. monocytogenes isolated from Guinea pig , TEM , 38,867 bp , Siphoviridae , Control of L-forms of L. monocytogenes on surfaces , [ , ] .

Techniques: Bacteria, Control, Isolation, Activity Assay, Inhibition, Recombinant

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Global distribution of a toxin-producing bacterial-fungal symbiosis. (A) Symbiotic bacteria ( Mycetohabitans sp.) residing within the fungal hypha of R. microsporus , produce a mixture of toxic secondary metabolites (rhizoxins). (B) Rhizoxin-producing Rhizopus - Mycetohabitans strains were isolated from environmental samples from geographically distinct sites covering all five continents. In one of the eight toxinogenic strains ( R. microsporus ATCC 62417, blue), rhizoxin causes blight disease in rice seedlings, while the ecological role of rhizoxin in the other, nonpathogenic Rhizopus strains is currently unknown.

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Bacteria, Isolation, Environmental Sampling

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Predation of Protostelium aurantium on spores of R. microsporus . (A) Fluorescence microscopy images showing FITC-stained, dormant R. microsporus spores (top) and ingestion of a swollen R. microsporus spore by P. aurantium (bottom). Scale bars, 5 μm. (B) Feeding of P. aurantium on dormant spores (top) leads to a reduced survival rate of P. aurantium compared to swollen spores (bottom). n = 3 independent replicated experiments ± 1 SEM. One-way ANOVA was performed with Tukey’s multiple-comparison test (*, P < 0.05; see also Table S1B ).

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Fluorescence, Microscopy, Staining, Comparison

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Culture extracts from symbiotic Rhizopus microsporus kills Protostelium aurantium . (A) The survival of P. aurantium , indicated by the diameter of the predation plaque (clearance of yeast), is significantly reduced in cultures that were exposed to 2% crude culture extract from symbiotic R. microsporus (RMsym). Incubation with solvent alone (DMSO) or apo-symbiotic R. microsporus (RMapo) has no effect on the viability of P. aurantium. Circles indicate independent replicated experiments ( n = 3) ± 1 SEM (gray bars). One-way ANOVA with Tukey’s multiple-comparison test was performed (*, P < 0.0001; see Table S2 ). (B) Photographs of yeast agar plates showing the predation plaque by P. aurantium (arrowheads). (C) HPLC profiles of crude extracts from symbiotic and endosymbiont-free R. microsporus showing a mixture of rhizoxin derivatives, including the two major bacterial rhizoxin congeners (rhizoxin S1 and rhizoxin S2). The peak correlating to rhizoxin is marked with an asterisk (*). Monitoring was done at 310 nm (see Fig. S2 ). The peak areas were integrated to calculate the concentration of rhizoxin S1 (1.2 μM) and rhizoxin S2 (1.7 μM), as well as all rhizoxin congeners combined (8.7 μM). (D) Fluorescence microscopy images of symbiotic R. microsporus and endosymbiont-free R. microsporus . Green fluorescence indicates presence of endosymbionts (SYTO9). Scale bars, 5 μm.

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Incubation, Solvent, Comparison, Concentration Assay, Fluorescence, Microscopy

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Inhibitory effects of crude extracts and pure rhizoxin S2 on C. elegans . (A) C. elegans , coincubated with E. coli OP50 cells as food source, were exposed to 2% crude culture extracts from symbiotic R. microsporus (RMsym), endosymbiont-free Rhizopus microsporus (RMapo), axenically grown endosymbiotic M. rhizoxinica HKI-0454 (labeled MR), Mycetohabitans endofungorum HKI-0456 (labeled ME), and rhizoxin-deficient M. rhizoxinica (Δ rhiG ), as well as pure rhizoxin S2 (rhi S2). Since the number of viable nematode worms in the suspension is directly related to the E. coli cell density, the OD 600 values were plotted as a percentage of the starting OD 600 . Incubation with 18 mM boric acid (positive control) kills most of the nematodes ( E. coli density of 80%), while exposure to crude culture extracts has a mild effect on C. elegans viability. Circles indicate independent replicated experiments ( n = 3) ± 1 SEM (gray bars). One-way ANOVA with Tukey’s multiple-comparison test was performed (*, P < 0.03; **, P < 0.002; ****, P < 0.0001; see Table S4 ). (B) Liquid feeding inhibition assay of C. elegans supplemented with the bacterial rhizoxin S2. Data points represent three independent replicated experiments ( n = 3) ± 1 SEM. Microscopic images of nematodes exposed to pure rhizoxin S2 are shown. Scale bars, 200 μm.

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Labeling, Suspension, Incubation, Positive Control, Comparison, Inhibition

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Feeding inhibition of A. avenae on R. microsporus . (A) A. avenae was coincubated with symbiotic R. microsporus (RMsym) or endosymbiont-free R. microsporus (RMapo) for 2 to 3 weeks. Nematode movement was recorded using a stereomicroscope with a frame rate of 1 fps. The liveliness of the worms was calculated from the ratio of the area covered by a worm, divided by the area of the worm itself, and scaled to the full length of the movie. The minimum scaled liveliness ratio (LR) for a live worm was set to 1.5, below this value the worm was declared inactive/dead. n = 3 independent replicated experiments ± 1 SEM. An unpaired t test with Welch’s correction was performed (*, P < 0.05; see Table S5 ). Microscope images of A. avenae used for analysis. Scale bars, 500 μm. (B) Illustrations of the LR at high (top) and medium (bottom) values. (Top) The worm shown in orange covers the red footprint area during the time course of the experiment. These images show the first (left column), middle (middle column), and final (right column) time points of the movie. The activity of a worm was characterized by dividing the endpoint footprint by the area of the worm at each time point. The resulting LR was 11.5 for the worm in the top row, thus indicating a very active nematode. (Bottom) A less active worm (green area) covered a smaller footprint (orange area), as shown by the LR value of 4.0. Scale bars, 300 μm. See the live videos of the segmented worms and their footprints in Videos S11 and S12 ( https://doi.org/10.5281/zenodo.6827988 ) for the worms with LR = 11.5 and LR = 4.0, respectively. (C) Time-lapse images of A. avenae feeding on endosymbiont-free R. microsporus (black circle). Endosymbiont-free R. microsporus ATCC 62417/S was coincubated with A. avenae for 24 h in a microchannel slide (Ibidi), and feeding was recorded on a spinning disc microscope (see ). Scale bars, 20 μm. No feeding was observed in worms that were coincubated with symbiotic R. microsporus (see Fig. S1D ; see also Video S2 [ https://doi.org/10.5281/zenodo.6827988 ]). (D) Microscopic images of A. avenae after exposure to different concentrations of pure rhizoxin S2 (rhi S2). Worms were healthy and alive when exposed to the solvent control (DMSO). Exposure to 114 μM ivermectin killed all worms. See the live videos of nematode movement in Videos S3 to S7 ( https://doi.org/10.5281/zenodo.6827988 ). Scale bars, 200 μm.

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Inhibition, Microscopy, Activity Assay, Solvent, Control

Journal: mBio

Article Title: Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

doi: 10.1128/mbio.01440-22

Figure Lengend Snippet: Schematic model of the ecological role of rhizoxin-producing endofungal bacteria ( M. rhizoxinica ). The fungal host ( Rhizopus microsporus ) utilizes the bacterial secondary metabolite rhizoxin to fend off fungivorous micropredators such as amoeba and nematodes. The absence of endofungal bacteria leads to R. microsporus being attacked and subsequently killed by protozoan and metazoan predators. The establishment of the Rhizopus - Mycetohabitans symbiosis may have first developed to provide protection against fungal predators, with the emergence of plant pathogenicity developing later.

Article Snippet: Both R. microsporus strains (ATCC 62417 and ATCC 62417/S) were cultivated on potato dextrose agar (PDA; Becton, Dickinson, & Company, Sparks, MD) at 30°C.

Techniques: Bacteria

Journal: Ecotoxicology and environmental safety

Article Title: Glutathione ameliorates the meiotic defects of copper exposed ovine oocytes via inhibiting the mitochondrial dysfunctions.

doi: 10.1016/j.ecoenv.2023.114530

Figure Lengend Snippet: Fig. 4. Effect of GSH supplementation on the mitochondrial dysfunctions related oxidative stress in the CuSO4 exposed ovine MII oocytes. A: Representative staining results of DCFH-DA. Note: NC, CuSO4 and GSH represent the normal control, CuSO4 and CuSO4 supplemented with GSH groups. DCFH-DA represents the DCFH-DA staining specific for the ROS production levels in ovine MII oocytes. DIC represents the bright field view of ovine MII oocytes. Merge represents the merged result of DCFH-DA staining and DIC microscopy. Scale bar= 50 µm. B: Relative staining intensity of DCFH-DA. Note: NC, CuSO4 and GSH represent the normal control group (n = 30), CuSO4 group (n = 30) and CuSO4 supplemented with GSH group (n = 30). Experimental data were analyzed by a one-way ANOVA with Post-hoc com parison and p < 0.05 was accepted as significant. Different lowercase letter in each column indicates significant differences between the corresponding experimental groups (p < 0.05). C: Representative staining results of MitoTracker. Note: NC, CuSO4 and GSH represent the normal control, CuSO4 and CuSO4 supplemented with GSH groups. MitoTracker represents the MitoTracker staining specific for the mitochondrial activities in ovine MII oocytes. DIC represents the bright field view of ovine MII oocytes. Merge represents the merged result of MitoTracker staining and DIC microscopy. Scale bar= 50 µm. D: Relative staining intensity of MitoTracker. Note: NC, CuSO4 and GSH represent the normal control group (n = 30), CuSO4 group (n = 30) and CuSO4 supplemented with GSH group (n = 30). Experimental data were analyzed by a one-way ANOVA with Post-hoc comparison and p < 0.05 was accepted as significant. Different lowercase letter in each column indicates sig nificant differences between the corresponding experimental groups (p < 0.05). E: Representative IF staining results of FDX1. Note: NC, CuSO4 and GSH represent the normal control, CuSO4 and CuSO4 supplemented with GSH groups. FDX1 represents the representative IF staining results of FDX1 in ovine MII oocytes. DIC rep resents the bright field view of ovine MII oocytes. Merge represents the merged result of FDX1 and DIC microscopy. Scale bar= 50 µm. F: Relative IF staining intensity of FDX1. Note: NC, CuSO4 and GSH represent the normal control group (n = 30), CuSO4 group (n = 30) and CuSO4 supplemented with GSH group (n = 30). Experimental data were analyzed by a one-way ANOVA with Post-hoc comparison and p < 0.05 was accepted as significant. Different lowercase letter in each column indicates significant differences between the corresponding experimental groups (p < 0.05). G: Representative IF staining results of GPX4. Note: NC, CuSO4 and GSH represent the normal control, CuSO4 and CuSO4 supplemented with GSH groups. GPX4 represents the representative IF staining results of GPX4 in ovine MII oocytes. DIC represents the bright field view of ovine MII oocytes. Merge represents the merged result of GPX4 and DIC microscopy. Scale bar= 50 µm. H: Relative IF staining intensity of GPX4. Note: NC, CuSO4 and GSH represent the normal control group (n = 30), CuSO4 group (n = 30) and CuSO4 supplemented with GSH group (n = 30). Experimental data were analyzed by a one-way ANOVA with Post-hoc comparison and p < 0.05 was accepted as significant. Different lowercase letter in each column indicates significant differences between the corresponding experimental groups (p < 0.05).

Article Snippet: For IF staining, MII oocytes after PFA fixation, Triton X-100 permeabilization and BSA blocking were collected and incubated with a rabbit monoclonal anti-γH2A antibody (with 1:200 dilutions in 1% BSA solution, ab81299, Abcam, Shanghai, China), a rabbit polyclonal anti- J. Ren et al. Ecotoxicology and Environmental Safety 251 (2023) 114530 FDX1 antibody (with 1:200 dilutions in 1% BSA solution, 12592–1-AP, Proteintech, Wuhan, China) and a mouse monoclonal anti-GPX4 antibody (with 1:200 dilutions in DPBS solution, 67763–1-Ig, Proteintech, Wuhan, China) at 4 ◦C overnight.

Techniques: Staining, Control, Microscopy, Comparison