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Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity

• Authors : Arunsan, Patpicha; Ittiprasert, Wannaporn; Smout, Michael J ...

• Source: eLife ; volume 8 ; ISSN 2050-084X

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Figure 7—figure supplement 1. TLR4 signals within microglial cells to limit morbidity in response to JHMV infection of the CNS. ; (A) Brains from R26R-EYFP; nestin-Cre mice were gated on YFP+ (left) or YFP- (right) populations and microglial markers displayed. (B) qPCR of TLR4 from unpurified splenocytes, CD11b+ MACS purified splenocytes, and CD11b+ MACS purified brain cells of tamoxifen-treated control (TLR4-fl/fl) and microglialΔTLR4 (CX3CR1CreER); TLR4-fl/fl) mice. Data represented as mean ± SEM. Means are calculated from biological replicates.

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Figure 6—figure supplement 1. TLR4 signals within microglial cells to limit morbidity in response to JHMV infection of the CNS. ; (A) Clinical scores (left) and area under the curve (right) of TLR2 KO vs WT mice after infection with JHMV (n = 7–8 ). (B) Number of MHCII-positive macrophages from WT and TLR4-/- mice under steady state conditions (n = 5). (C) HEK-Blue TLR4 detection cell lines were incubated with indicated concentrations of JHMV to determine whether JHMV itself could signal through TLR4, PBS or 100 ng/ml LPS. Data points indicate technical replicates (n = 3) and mean ± SEM. Data represented as mean ± SEM. Means are calculated from biological replicates.

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Figure 7—source data 1. Accelerated morbidity conferred by double deficiency of ATG16L1 and ZBP1 in myeloid cells following LPS-mediated sepsis in mice.

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Figure 1. lncRNA loci are recurrently mutated in patients with neurodevelopmental disorders. ; (A) Schematic representation of CNV morbidity map analysis for candidate lncRNAs and all other iN lncRNAs loci. The 35 mouse lncRNA candidates (28 human loci) is from Figure 1—figure supplement 1H. (B) Top: Representative tracks for lncRNA E locus, also known as lnc-NR2F1. Depicted in blue are deletions and in red duplications. Arrow points to patient with focal deletion affecting the lnc-NR2F1 locus only. Bottom: Custom CGH arrays used to validate chromosomal aberration in patient 9900850 harboring focal deletion represented in green signal. (C) Genetic pedigree analysis for family with paternally inherited balanced chromosomal translocation (5;12) (q15;q15), including a summary of clinical features for patient CMS12200 and father. The mother has a normal karyotype. Listed in the box are the symptoms of the patients. (D) Top: Circa plot representing the pathogenic chromosomal event for patient CMS12200 involving chromosomes 5 and 12. Bottom: Representative chromosome ideogram and track of the balanced chromosomal break affecting patient CMS12200. Below the ideoplot is the schematic representation of predominant human isoforms for lnc-NR2F1 and the site of the break site disrupting the long isoforms. (E) The locations of the probes are in Figure 1—figure supplement 4C. Left: Metaphase spread from patient CMS12200 with the t(5;12) translocation showing FISH signals obtained with the clone RP11-608G16 (green) spanning Chromosome five breakpoint, and a Chromosome five telomere-specific probe (red). Middle: Metaphase spread from patient CMS12200 with the t(5;12) translocation showing FISH signals obtained with the clone RP11-597C7(green) proximal to Chromosome 12 breakpoint, and a Chromosome 12 centromere-specific probe (red). Right: Metaphase spread from patient CMS12200 with the t(5;12) translocation showing FISH signals obtained with the clone RP11-641O3 (green) distal to Chromosome 12 breakpoint, and a Chromosome 12 centromere-specific probe (red).

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Figure 7—figure supplement 1. Accelerated morbidity conferred by double deficiency of ATG16L1 and ZBP1 in myeloid cells following LPS-mediated sepsis in mice. ; (A) Statistical analysis of Kaplan-Meier curve depicted in Figure 7. P-values are generated using log-rank test. (B) Inflammatory cytokines, death ligands and TLR ligands induce necroptotic signaling upon caspase-inhibition. Autophagy promotes turnover of TRIF, RIPK1 and RIPK3 to control necroptosis in healthy macrophages. (C) In the absence of autophagy, accumulation of active TRIF, RIPK1 and RIPK3 enhances necroptosis as well as inflammatory cytokine production. Accumulation of ZBP1 attenuates TRIF-mediated necroptosis during autophagy deficiency. During TLR3- or TLR4- activation, overabundance of TRIF drives RIPK3-dependent necroptosis that is resistant to RIPK1 inhibition. Autocrine signaling via non-TRIF TLRs may contribute to enhanced necroptosis. Dotted lines depict indirect signaling events; solid lines depict direct signaling events.

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Decision letter: Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity

• Authors : Lok, James B; Garrett, Wendy S

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Author response: Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity

• Authors : Arunsan, Patpicha; Ittiprasert, Wannaporn; Smout, Michael J

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Figure 6. Endogenous RAE-1ε - NKG2D interactions limit NK responses to tumors. ; (A) WT or NKG2D-KO mice (n = 29–30) were challenged with 2 × 104 B16 cells i.v. and monitored for morbidity. Matched groups of each strain (n = 9–10) were depleted of NK cells before implanting the tumor cells. Data are combined results of three independent experiments. (B) WT or NKG2D-KO mice (n = 9) were challenged with 5 × 103 B16 cells s.c. and monitored for tumor growth. Data are representative of three independent experiments. (C) WT or NKG2D-KO mice (n = 9) were challenged with 5 × 105 RMA-S cells s.c. and monitored for tumor growth. Data are representative of three independent experiments. (D) WT or NKG2D-KO mice (n = 9) were challenged with 5 × 104 B16-MULT1 cells s.c. and monitored for tumor growth. Data are representative of two independent experiments. (E) WT or RAE-1-KO mice were challenged with 5 × 104 B16-MULT1 cells s.c. and monitored for tumor growth. (n = 12) Data are representative of three independent experiments. (F) WT or RAE-1-KO mice (n = 7–8) were challenged with 2 × 106 TRAMP-C2 cells s.c. and monitored for tumor growth. Data are representative of three independent experiments. Statistical significance was determined using two-way ANOVA. Data represent means ± SEM.

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Figure 1—figure supplement 3. Stable neuronal function and environment-specific activity. ; (A,B) Both ‘activity divergence’, the difference in event rate between trials in different environments relative to the difference in event rate between trials in the same environment (A), and ‘peak displacement’, the average distance between locations of peak activity in different environments (B), did not change over the course of the experiment (One-way repeated-measures ANOVA; F(1.9, 7.6) = 1.06, p = 0.39 and F(2.6, 10.5) = 1.22, p = 0.345 respectively). (C) Average number of Ca2+ events detected on different days of the experiment did not change with time (One-way repeated-measures; F(1.87, 7.49) = 1.19, p = 0.35). (D) Amplitudes of Ca2+ events remained similar over the course of the experiment (One-way repeated-measures; F(1.32, 5.29) = 3.29, p = 0.35.). (E) Fractions of place cells from the population of cells active in each environment were similar for both environments and did not change with time. Two-way repeated measures ANOVA between environment A and B (F < 1, p = 0.42), over time (F(2.32,9.30) = 1.99, p = 0.19), and environment X time interaction (F(1.93, 7.72) = 1.71, p = 0.243). Data are mean ± s.e.m. (F) Representative two-photon microendoscopy images of CA1 neurons performed before and after the experiment. The images revealed no increase in the number of cells with filled nuclei (a marker for GCaMP-induced neuronal morbidity). Scale bar is 50 μm.

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