Journal: Marine Life Science & Technology

Article Title: Emerging role of lipid droplets in obscure puffer immune response against Vibrio harveyi

doi: 10.1007/s42995-025-00286-w

Figure Lengend Snippet: Increasing cellular LD numbers in TEM images of Vibrio harveyi stimulated liver. A, D TEM images revealed fewer LDs in the liver from control group at 24 h ( A ) and 48 h ( D ), respectively; B, C TEM images revealed increased cellular LD numbers in the liver at 24 h post V. harveyi stimulation ( B ), and the boxed area was enlarged in the ( C ). D, E TEM images revealed increased cellular LD numbers and size in the liver at 48 h post V. harveyi stimulation ( D ), and the boxed area was enlarged in the ( E ). Scale bar = 1 μm ( A, B, D, E ); Scale bar = 500 nm ( C, F )

Article Snippet: For the second group, each fish was pretreated with 100 μL of V. harveyi and T863 (2.5 μmol/L, MCE, USA) was added 1 h later.

Techniques: Control

Journal: Marine Life Science & Technology

Article Title: Emerging role of lipid droplets in obscure puffer immune response against Vibrio harveyi

doi: 10.1007/s42995-025-00286-w

Figure Lengend Snippet: Vibrio harveyi stimulation induced lipid droplets’ formation by promoting the synthesis of neutral lipids. A The contents of triglycerides (TAGs) in liver and kidneys at 24 h post V. harveyi stimulation. B The contents of cholesterol esters (CEs) in liver and kidneys at 24 h post V. harveyi stimulation. C–K qRT-PCR analysis of expression of genes involved the synthesis of neutral lipids (DGAT1, DGAT2, ACSL3, FASN, ACAT, FATP4, CD36, ATGL, and ATP5D) in control, V. harveyi and killed V. harveyi stimulated groups. Data are mean ± SD, n = 3 biological replicates. Two-tailed unpaired t tests. ** P < 0.001; * P < 0. 05

Article Snippet: For the second group, each fish was pretreated with 100 μL of V. harveyi and T863 (2.5 μmol/L, MCE, USA) was added 1 h later.

Techniques: Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: Marine Life Science & Technology

Article Title: Emerging role of lipid droplets in obscure puffer immune response against Vibrio harveyi

doi: 10.1007/s42995-025-00286-w

Figure Lengend Snippet: The antibacterial capacity assay of LD proteins. A Vibrio harveyi were cultured for indicated times in either growth media with Tris-HCL (red) or in media containing 175 μg of proteins from control LDs (blue), or V. harveyi stimulated LDs (black). At indicated times, the OD600 of V. harveyi growth was measured and quantified relative to the control group. B Staphylococcus aureus were cultured for indicated times in either growth media with Tris-HCL (red) or in media containing 175 μg of proteins from control LDs (blue), or V. harveyi stimulated LDs (black). At indicated times, the OD600 of S. aureus growth was measured and quantified relative to the control group. C, D After 2 h, serial dilutions of the culture were plated in soft agar, and the number of growing colonies (colony-forming units, CFU) was counted and referred to the control condition (representative of three fish per condition). Data are mean ± SD, n = 3 biological replicates. Two-tailed unpaired t tests. ** P < 0.001; * P < 0. 05

Article Snippet: For the second group, each fish was pretreated with 100 μL of V. harveyi and T863 (2.5 μmol/L, MCE, USA) was added 1 h later.

Techniques: Cell Culture, Control, Two Tailed Test

Journal: Marine Life Science & Technology

Article Title: Emerging role of lipid droplets in obscure puffer immune response against Vibrio harveyi

doi: 10.1007/s42995-025-00286-w

Figure Lengend Snippet: Quantitative proteomics revealed dynamic changes of LD proteins post Vibrio harveyi stimulation. A The principal component analysis (PCA) at the proteomics level for a total of six liver LD samples of the control and Vh groups. B Differentially expressed proteins were displayed by volcano plots. The vertical lines correspond to Log 10 P value, and the horizontal line represents the Log 2 E/C Ratio. Upregulation of protein expression in Vh group is shown in red, while downregulation is shown in blue. Unchanged proteins were indicated as gray. C Subcellular localization analysis of differentially expressed proteins. The pink indicated the upregulated proteins, while the blue indicated the downregulated proteins. D KOG classifications of differentially expressed proteins. The ordinate denotes the number of proteins in each KOG category

Article Snippet: For the second group, each fish was pretreated with 100 μL of V. harveyi and T863 (2.5 μmol/L, MCE, USA) was added 1 h later.

Techniques: Quantitative Proteomics, Control, Expressing

Journal: Marine Life Science & Technology

Article Title: Emerging role of lipid droplets in obscure puffer immune response against Vibrio harveyi

doi: 10.1007/s42995-025-00286-w

Figure Lengend Snippet: The inhibition of LDs formation decreases PLIN3 expressions, TAG contents, and mRNA levels of immune-related genes. A Treatment with DGAT1 inhibitor T863 significantly decreased triglycerides (TAGs) contents in liver under the higher dose (0. 25 mg). B Treatment with DGAT1 inhibitor T863 decreased LD-resident protein PLIN3 expression in liver both under the lower dose (0.125 mg) and higher dose (0. 25 mg). C Pre-treatment with DGAT1 inhibitor T863 reduced the contents of liver triglycerides (TAGs) at 24 h in liver post Vibrio harveyi stimulation. D Pre-treatment with DGAT1 inhibitor T863 attenuated the expressions of LD-resident protein PLIN3 at 24 h in liver post V. harveyi stimulation. E, F Pre-treatment with DGAT1 inhibitor T863 significantly downregulated the mRNA levels of immune-related genes in liver. Scale bar = 1 μm ( A, B, D, E ); Scale bar = 500 nm ( C, F )

Article Snippet: For the second group, each fish was pretreated with 100 μL of V. harveyi and T863 (2.5 μmol/L, MCE, USA) was added 1 h later.

Techniques: Inhibition, Expressing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: MitoTracker staining in T863-treated Toxoplasma. (A and B) Fluorescence microscopy of HFF infected with GFP-expressing Toxoplasma, staining for mitochondria using MitoTracker (MitoT). (A) Infected cells were incubated with T863 at the indicated concentrations for 20 to 22 h, and the cells were compared to cells treated with 10 μM pyrimethamine (PYR) as a negative control (−ve ctl) or the DMSO control. (B) Uninfected cells were treated with 40 μM T863 or the DMSO control strained with MitoTracker. (C) Quantification of the MitoTracker staining of the cells in the experiment whose results are presented in panel A by counting the number of PV containing parasites positively labeled with the mitochondrial probe detecting live cell/metabolically active mitochondria in infected cells. The positive control (+ve ctl) corresponds to conditions without any addition to the culture medium. The percentage of viable PV is graphed. P values were determined by Student's t test. *, P < 0.0095; **, P < 0.0003; ***, P < 0.00003.

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Staining, Fluorescence, Microscopy, Infection, Expressing, Incubation, Negative Control, Control, Labeling, Metabolic Labelling, Positive Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: Effect of T863 on Toxoplasma replication, morphology, and lipid body abundance. (A) Toxoplasma replication assessed by PV size at 24 h p.i. in the presence of T863 at the indicated concentrations. The number of parasites per PV in Toxoplasma-infected HFF is displayed as the percentage of PV with that number of parasites among all PV. Data show the means ± SD from at least 20 randomly selected vacuoles from 3 independent experiments. P values were determined by Student's t test. *, P < 0.009; **, P < 0.00001. The results of trypan blue exclusion tests performed in replicates for evaluating the viability of HFF exposed to T863 for 24 h were 97.5% viable cells under the control condition (DMSO) and 97.2% with 80 μM T863. (B) Predicted doubling times of parasites grown in the presence of T863, calculated from the data acquired for panel a using the Predictive Replication Analysis algorithm. (C) Fluorescence microscopy of HFF infected with RFP-expressing Toxoplasma incubated for 24 h with T863 at different concentrations showing differences in PV size and misshapen parasites in T863-treated cells with reduced BODIPY 493/503 signals. Arrowheads pinpoint empty structures. (D) IFA on intracellular Toxoplasma incubated with 80 μM T863 for 24 h and immunostained with anti-GRA7 antibody, confirming that the empty structures were PV. (E) Quantification of Toxoplasma replication in HFF at 24 h p.i., assessed by the use of tritiated uracil incorporation in the presence of 80 μM T863 for 24 h. Data are percentages ± SD relative to the value for the control (DMSO; the value for which was set as 100%) for 4 biological replicates with experimental triplicates. The absolute values were as follows: for experiment 1, 86,951 ± 5,730 cpm for DMSO and 1,604 ± 94 cpm for T863; for experiment 2, 85,902 ± 10,896 cpm for DMSO and 1,745 ± 553 cpm for T863; for experiment 3, 10,836 ± 496 cpm for DMSO and 995 ± 112 cpm for T863. *, P < 0.0001, Student's t test. (F) Fluorescence microscopy of D1D2KO MEF infected with RFP-expressing Toxoplasma incubated for 24 h with 15 μM T863 and stained with BODIPY 493/503 (green).

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Infection, Control, Fluorescence, Microscopy, Expressing, Incubation, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: Lipid body abundance in Toxoplasma exposed to T863 and supplemental OA. (A) Fluorescence microscopy of HFF infected with RFP-expressing Toxoplasma incubated with excess OA and/or 15 μM T863 for 24 h and stained with BODIPY 493/503 (green). (B) Quantification of the BODIPY 493/503 signal in Toxoplasma cultivated as described in the legend to panel A. The graph shows the average LD volume, calculated using Volocity software, from BODIPY 493/503 staining. Data (mean volumes ± SD) acquired from three independent experiments. P values were determined by Dunnett's test. *, P < 0.0082; **, P < 0.0001.

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Fluorescence, Microscopy, Infection, Expressing, Incubation, Staining, Software

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: Ultrastructure of Toxoplasma incubated with T863. (A to E) Transmission EM of Toxoplasma-infected HFF incubated without drug or with T863 at 7.5 or 15 μM for 24 h showing cytopathies, such as ER swelling and membranous tubules (yellow arrowheads). No cytopathies were observed in the host cell (hc). a, apicoplast; DG, dense granule; Go, Golgi apparatus; m, mitochondrion; mi, micronemes; n, nucleus; NE, nuclear envelope; RB, residual body. Bars, 300 nm.

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Incubation, Transmission Assay, Infection

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: Effect of T863 on differentiated bradyzoites in culture. (A) (Top) Schema outlining our experimental protocol. (a to f) Fluorescence microscopy of encysted ME49 bradyzoites differentiated for 3 days before staining with TRITC-lectin, DAPI, and BAG1 as a marker of the bradyzoite plasma membrane. At 40 to 42 h postinfection, 80 μM T863 or DMSO (control) was added to the BCM. Following treatment for 2 days, large and small cysts filled with parasites are detected (a and b) and cysts with misshapen parasites are observed under drug treatment (c to f). The images shown have the same magnification. Bars, 17 μm. (B) Quantification of cyst size upon T863 treatment, as described in the legend to panel A. (a) Box plot graphs from 3 independent experiments (40 to 46 cysts per experiment) showing cyst volumes, calculated using Volocity software, where cysts are detected on the basis of TRITC-lectin fluorescence, as described in Materials and Methods. P values were determined by a 2-tailed t test type 3. (b) Graph combining data from the 3 assays whose results are presented in panel a, representing the averaged cyst volume expressed as a percentage of that obtained with the DMSO control (means ± SD; P values were determined by 2-tailed t test type 3).

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Fluorescence, Microscopy, Staining, Marker, Clinical Proteomics, Membrane, Control, Software

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Novel Approaches To Kill Toxoplasma gondii by Exploiting the Uncontrolled Uptake of Unsaturated Fatty Acids and Vulnerability to Lipid Storage Inhibition of the Parasite

doi: 10.1128/AAC.00347-18

Figure Lengend Snippet: Effect of T863 on LD in differentiated bradyzoites in culture. (A) (Top) Schema outlining our experimental protocol. (a to c) Fluorescence microscopy of encysted ME49 bradyzoites before staining with BODIPY 493/503, TRITC-lectin, and DAPI, revealing numerous LD in control cysts (a), while no LD (b) or rare LD (c) were detected in T863-treated parasites. Parasite LD are intracytoplasmic, as evidenced in the orthogonal views (arrows). The images shown have the same magnification. Bars, 17 μm (z-slices) and 19 μm (orthogonal views). (B) Quantification of the BODIPY 493/503 signal in differentiated cysts upon T863 treatment. (a) Box plot graphs from 3 independent experiments (41 to 46 cysts per experiment) showing the sum of BODIPY 493/503 fluorescence per cyst volume corrected for the background as described in Materials and Methods. P values were determined by a 2-tailed t test type 3. (b) Graph combining data from the 3 assays whose results are presented in panel a, representing the averaged sum of the BODIPY 493/503 fluorescence normalized to the cyst volume and corrected for the background (means ± SD; P values were determined by 2-tailed t test type 3).

Article Snippet: T863 { trans -4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexane acetic acid} was obtained from AstraZeneca (Cambridge, UK).

Techniques: Fluorescence, Microscopy, Staining, Control