Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: In the PHF → Ab dosing paradigm, 1D9 IgG1 and IgG2A are the most efficacious subclasses in preventing PHF-induced toxicity and seeding. All 2B8 IgG subclasses are toxic . Mixed JNPL3 cultures were incubated with 10 μg/ml PHF followed 24 h later by 10 µg/ml IgG subclasses (n = at least 6 replicates per condition). Cells were collected 96 h after the antibody application. a. In the 1D9 group, PHF alone increased LDH levels and 1D9 IgG2B had no effect on PHF toxicity (177 and 208 % of control values, p = 0.02 and 0.0008, overall p = 0.0002, one-way ANOVA). In contrast, 1D9 IgG1 prevented PHF toxicity (108% of control, p = 0.05). b. In the 2B8 group, there was an overall treatment effect (p < 0.0001, one-way ANOVA). 2B8 IgG1, IgG2A and IgG2B samples had increased LDH relative to untreated samples (187, 214, and 248% of control values, p = 0.009, = 0.0002, and < 0.0001), and IgG2A and IgG2B treated cells were also elevated compared to PHF alone (p = 0.05, 0.0004). c. Immunoblots showing NeuN levels in the control and treated JNPL3 cultures. d. In the 1D9 group, PHF alone decreased NeuN (43% of control, p < 0.0001, overall p < 0.0001, one-way ANOVA). 1D9 IgG1, IgG2A, and IgG2B prevented this toxicity (NeuN levels 86, 100, and 65% of control values, p < 0.0001, < 0.0001, and 0.04 respectively). e. In the 2B8 group, PHF alone was toxic as measured by NeuN levels (62% of control, p < 0.0001, overall p < 0.0001, one-way ANOVA). None of the 2B8 IgG subtypes prevented the PHF-induced toxicity and had reduced NeuN levels relative to untreated control cells (55, 77, 42, and 34% of control values, P < 0.0001, 0.04, < 0.0001, < 0.0001). f. Immunoblots from the same JNPL3 cell lysate used in the toxicity experiments were probed for total tau. g. In the 1D9 group, PHF alone increased intracellular tau relative to untreated control (ratio tau/NeuN 1.96, p < 0.0001, overall p < 0.0001, one-way ANOVA). 1D9 IgG1, IgG2A and IgG2B prevented tau seeding (ratio tau/NeuN 1.05, 0.97, 1.39, p < 0.0001 compared to PHF alone for all). h. In the 2B8 group, PHF alone increased total tau in the cells (ratio tau/NeuN 1.47, p = 0.016, overall p = 0.0003, one-way ANOVA). Cells treated with 2B8 IgG1, IgG2B and IgG3 also had higher total tau relative to untreated control (ratio tau/NeuN 1.55, 1.68 and 1.61, p = 0.013, 0.0015, and 0.013), and none of the antibodies prevented the PHF-induced tau seeding. i. The same samples were used in immunoblotting to measure tau phosphorylated at Ser199. j. In the 1D9 group, PHF increased intracellular phospho-tau (pSer199/NeuN ratio 2.76, p < 0.0001, overall p < 0.0001, one-way ANOVA). 1D9 IgG1, IgG2A and IgG2B prevented this increase (ratio pSer199/NeuN 1.31, 1.03, and 1.86, p < 0.0001, < 0.0001, 0.03). k. In the 2B8 group, PHF alone increased phospho-tau seeding (ratio pSer199/NeuN 1.93, p = 0.001, overall p < 0.0001, one-way ANOVA). Cells treated with 2B8 IgG1, IgG2A and IgG3 also had an increase in pSer199 relative to the untreated controls (ratio pSer199/NeuN 1.83, 2.11, 2.76, p = 0.013, 0.0005, < 0.0001), with none of the subtypes resulting in different pSer199 levels relative to PHF alone. Bars represent average ± SEM. # p ≤ 0.05, ## p ≤ 0.001, ### p ≤ 0.001, #### p < 0.0001, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p < 0.0001. # significant compared to untreated control, * significant compared to PHF alone.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Incubation, Control, Western Blot

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: 1D9 and 2B8 IgG subclasses bind recombinant-, phosphorylated-, and PHF-tau in solution phase BLI assay . Whole IgG subclasses (Fc-(sdAb) 2 ) were loaded onto Anti-Mouse IgG Fc Capture (AMC) biosensors. K D values were determined using increasing concentration of recombinant- (rec-tau), phosphorylated- (p-tau), and PHF-tau. The IgG subclasses displayed an apparent increased affinity for tau relative to their sdAbs. This results from increased avidity because of bivalent target engagement. a-c. 1D9 IgG1, IgG2A, and IgG2B had 9.6, 7.8-, and 3.9-fold stronger avidity for rec-tau compared to 1D9 sdAb (K D = 4.2 nM, 5.1 nM, and 10.3 nM, respectively). d-f. 2B8 IgG1, IgG2A, and IgG2B had 4.7-, 4.2- and 5.8-fold higher avidity than the sdAb for rec-tau (K D = 3.0 nM, 3.4 nM, and 2.5 nM, respectively). g-i. For p-tau, whole 1D9 IgGs had 3 – 3.6 fold higher avidity relative to their sdAb (K D = 9.1 nM, 11.0 nM, and 10.0 nM for IgG1, IgG2A, and IgG2B, respectively). j-l. The effect of bivalent engagement was also seen with 2B8 in assays with p-tau as IgG1 (8.3 nM), IgG2A (6.8 nM), and IgG2B (11.2 nM) had 4.0-, 4.8-, and 2.9-fold stronger avidity compared to the 2B8 sdAb. m-o. When tested against human derived PHF, 1D9 IgG1, IgG2A, and IgG2B exhibited 2.8-, 3.0-, and 1.9-fold stronger avidity compared to 1D9 sdAb (K D = 8.9 nM, 8.4 nM, and 13.5 nM, respectively). p-r. K D values for 2B8 IgG subclasses were also higher for PHF than their sdAb (14.2 nM, 69.6 nM, and 51.0 nM, for IgG1, IgG2A and IgG2B, respectively). * Due to low Fc binding IgG3 subtypes did not yield results in these tests.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Recombinant, Concentration Assay, Derivative Assay, Binding Assay

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: 1D9 and 2B8 IgG subclasses bind recombinant tau in solid phase BLI assay; 1D9 IgG2B has highest affinity of the subclasses for tau in solid phase ELISA. Only the IgG3 subclass of 1D9 does not bind to PHF-tau in solution. a-h. In the BLI experiments, biosensors were loaded with biotinylated recombinant tau (rec-tau). Association and dissociation of increasing concentrations of whole IgG subtypes were measured and K D values determined. Interestingly, there was no major increase if any in avidity over the unmodified 1D9 and 2B8 sdAbs despite the presence of two binding sites on the whole IgG. a-d. 1D9 subtypes IgG1, IgG2A, IgG2B, and IgG3 had similar K D values (26.0 nM, 39.1 nM, 33.1 nM, and 55.5 nM) that were comparable to 1D9 sdAb (K D = 50.8 nM, see j). e-h. 2B8 subtypes also had similar K D values (37.0 nM, 47.3 nM, 51.9 nM, and 53.3 nM for 2B8 IgG1, IgG2A, IgG2B, and IgG3) that were comparable to 2B8 sdAb (K D = 49.3 nM, see k). i-l. For the standard ELISA, plates were coated with solubilized PHF, sarkosyl insoluble, and sarkosyl soluble tau fractions (1 μg per well). Plates were blocked and then incubated for 3 h with serial dilutions of each 1D9 subtype. i. IgG2B had significantly higher binding than the other subclasses to PHF-tau from 1/40 through 1/25000 (p values from 0.03 to < 0.0001, two-way ANOVA). j. For sarkosyl insoluble tau, 1D9 IgG2B binding was significantly higher than the other subtypes from dilutions 1/40 through 1/5000 (p from 0.03 to < 0.0001, two-way ANOVA). k. On plates coated with sarkosyl soluble tau, 1D9 IgG2B had significantly higher binding at 1/40 (p < 0.0001 for all, two-way ANOVA) and at 1/200 (p = 0.0004 to < 0.0001 for all) and at 1/1000 (p = 0.013 and 0.004 relative to IgG2A and IgG3). l. For the competitive ELISA, plates were coated with solubilized PHF-tau and a single concentration of each of the 1D9 IgG subclass was pre-incubated with increasing concentrations of PHF-tau before adding it to the wells. 1D9 IgG1 had decreased binding to the plate at the two highest PHF concentrations (p = 0.03, < 0.0001, two-way ANOVA) compared to the no PHF control. Plate binding was significantly reduced for 1D9 IgG2A at the three highest PHF pre-incubation concentrations (p = 0.013, 0.004, 0.0014). IgG2B had decreased binding at the two highest PHF concentrations as well (p = 0.03, 0.012). There was no change with IgG3, indicating that it did not bind to PHF-tau in solution. Bars represent average ± SEM. * p ≤ 0.05, ** p ≤ 0.01, **** p < 0.0001, compared to 1D9 IgG2B in panels i-k, and relative to samples receiving no PHF pre-incubation in panel l.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Competitive ELISA, Concentration Assay, Control

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: 1D9 subclasses are not toxic in mixed neuron/glia cultures while 2B8 subclasses display toxicity that is dependent on the presence of pathological tau. a-e. Mixed neuron/glia cultures prepared from JNPL3 mice were incubated with 1D9 and 2B8 subtypes alone (10 μg/ml) for 96 h (n = at least 6 replicates per condition). a. There was no significant change in LDH levels in cultures treated with any of the 1D9 subtypes. b. Incubation with 2B8 IgG2A, IgG2B and IgG3 alone increased LDH relative to untreated controls (206, 196, and 176% of control values, p = 0.002, 0.006, 0.046, overall p = 0.003 one-way ANOVA). There was also a strong trend towards increased LDH with 2B8 IgG1 (p = 0.07) c. Immunoblots showing NeuN levels in treated JNPL3 cell lysates. d. None of the 1D9 subtypes altered NeuN levels relative to untreated controls. e. All 2B8 subtypes reduced NeuN levels compared to untreated cells (48, 48, 37, and 46 % of control for IgG1, 2A, 2B and 3, respectively, p ≤ 0.0001 for all, overall p < 0.0001 one-way ANOVA). f. Immunoblots showing NeuN levels in lysates of wild-type cells from mixed neuron/glia cultures treated with 2B8 sdAb. g-h. There were no significant changes in LDH levels (g) or NeuN levels (h) over the 6 day treatment period in the wild-type mouse cells, indicating that the toxic effects seen in the JNPL3 cells in b and e is dependent on pathological tau. Bars represent average ± SEM. # p ≤ 0.05, ### p ≤ 0.001, ##### p < 0.0001, compared to untreated control.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Incubation, Control, Western Blot

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: In the PHF + Ab paradigm, 1D9 IgG1 and IgG2A are the most efficacious subclasses in preventing PHF-induced toxicity and tau seeding, whereas all 2B8 IgG subclasses are toxic or ineffective . Mixed neuron/glia cultures prepared from JNPL3 pups were exposed to 10 µg/ml PHF and 10 μg/ml tau sdAb IgG subclasses added simultaneously (PHF + Ab, n = at least 6 replicates per condition). Cells were collected 96 h following treatment. a. For the 1D9 co-incubation samples, the PHF alone increased LDH levels in culture media (137% of control, p = 0.05, overall p = 0.006, one-way ANOVA). 1D9 IgG1, IgG2A and IgG2B prevented this increase but not 1D9 IgG3 (154% of control, p = 0.01). b. In the co-incubation condition, 2B8 IgG2A, IgG2B and IgG3 had higher LDH levels in the media compared to untreated samples, with IgG2B and IgG3 also significantly higher relative to PHF alone (279, 390, and 293% of control values, p = 0.001, 0.0001, and 0.0011 vs untreated control, p < 0.0001, and 0.04 vs PHF, overall p < 0.0001, one-way ANOVA). c. Immunoblots showing JNPL3 cell lysates probed for NeuN from samples treated with PHF and the IgG sdAb subclasses. d. In the 1D9 group, PHF alone decreased NeuN in cells (56% of control, p < 0.0001, overall p < 0.0001, one-way ANOVA). 1D9 IgG1 and IgG2A prevented PHF-induced toxicity (115 and 112% of control values, p < 0.0001 for both), while IgG2B and 3 did not. e. In the 2B8 group, PHF alone decreased NeuN (50% of control, p < 0.0001, overall p < 0.0001, one-way ANOVA). None of the 2B8 IgG subtypes prevented the PHF-induced toxicity (38, 25, 17, 25 % of control, p < 0.0001 for all). The 2B8 IgG2A, IgG2B, and IgG3 treated samples were also reduced compared to PHF alone (p = 0.03, 0.001, and 0.05). f. Tau immunoblots of the JNPL3 lysate from the same cultures used in the LDH and NeuN quantification. Tau values in g, h, j and k were normalized using NeuN to account for cell loss or retraction. g. In the 1D9 group, PHF alone increased intracellular tau (ratio tau/NeuN 1.94-fold over control, p = 0.0004, overall p < 0.0001, one-way ANOVA). 1D9 IgG1 and IgG2A prevented tau seeding resulting in lower intracellular tau levels relative to PHF alone (ratio tau/NeuN 0.87 and 0.75, p = 0.0016 and 0.0003) while IgG2B and IgG3 had no effect. h. In the 2B8 group, PHF alone increased intracellular tau (tau/NeuN ratio 1.91, p = 0.017, overall p = 0.0002, one-way ANOVA), and none of the 2B8 IgG subclasses prevented tau seeding. 2B8 IgG3 resulted in higher tau levels than in both untreated control and PHF alone cells (ratio tau/NeuN 3.81, p < 0.0001 and = 0.0093 respectively). i. The same JNPL3 lysate was also immunoblotted for tau phosphorylated at Ser199. j. In the 1D9 group, PHF increased intracellular phospho-tau relative to untreated control (ratio pSer199/NeuN 2.28, p = 0.0003, overall p < 0.0001, one-way ANOVA). Both 1D9 IgG1 and IgG2A prevented pathological tau seeding (ratio pSer199/NeuN ratio 0.84 and 0.86, p = 0.0012 and 0.0015). k. In the 2B8 group, IgG2A, IgG2B and IgG3 treated samples all contained higher levels of pSer199 tau than either untreated controls or cells exposed to PHF alone (ratio pSer199/NeuN 4.26, 4.57, and 4.50, p = 0.0005, 0.0001, and 0.0006 relative to untreated samples, p = 0.047, 0.013, and 0.040 relative to PHF alone, overall p < 0.0001, one-way ANOVA). Bars represent average ± SEM. # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001, #### p < 0.0001, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.00,1**** p < 0.0001. # significant compared to untreated control, * significant compared to PHF alone .

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Incubation, Control, Western Blot

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: 1D9 IgG3 has lower neuronal and microglial uptake relative to other IgG subclasses . Each 1D9 IgG subtype was labeled with CypHer5, a pH sensitive dye that fluoresces in acidic compartments such as the endosomal/lysosomal system. a. Mixed cortical JNPL3 cultures were incubated with 5 µg/ml of the labeled antibodies for one h without addition of exogenous PHF, after which live images were collected from each well (scale = 30 µm). b. The total number of pixels in each image (n = 18 images per group) that contained the antibody signal was determined, and 1D9 IgG3 showed significantly lower uptake compared to all three other subtypes (p < 0.0001 for all, overall p < 0.0001, one-way ANOVA). c. An additional group of cells was fixed and then stained with a total tau antibody to visualize neurons (scale = 20 µm). d. All 1D9 IgG subtypes showed a similar high degree of colocalization between the antibody and neurons (IgG1 75%, IgG2A 62%, IgG2B 65%, and IgG3 71%, n = 20 images per group), indicating that in the absence of exogenous tau, most antibody uptake is neuronal. e. CypHer 5 labeled 1D9 IgG subtypes (5 µg/ml) and human derived PHF-tau (1 μg/ml) were added to mixed neuron/glia cultures at the same time. Cultures were also incubated with fluorescent tomato lectin to label microglia. Live cell images of the cultures showed a high degree of antibody colocalization with glia for 1D9 IgG1, IgG2A and IgG2B after one h incubation (scale = 20 µm). f. Overall uptake in each image (n = 15 images per group) was determined, and 1D9 IgG3 had less uptake than either IgG1 or IgG2B (p = 0.001 and 0.002, overall p = 0.0002, one-way ANOVA). g. 1D9 IgG1, IgG2A and IgG2B showed a shift to a majority microglial uptake in the presence of PHF (82, 77, and 82%). IgG3 in contrast did not and its percentage of microglial uptake was lower relative to the other three (46% colocalization, p = 0.0015, 0.008, and 0.001 for IgG1, IgG2A, and IgG2B, overall p = 0.0003, one-way ANOVA). Bars represent average ± SEM. ** p ≤ 0.05, *** p ≤ 0.001, **** p < 0.0001.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: Labeling, Incubation, Staining, Derivative Assay

Journal: eBioMedicine

Article Title: Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity

doi: 10.1016/j.ebiom.2022.104249

Figure Lengend Snippet: 1D9 IgG1 and IgG2A improve clearance of interstitial tau relative to their sdAb in vivo, whereas the 2B8 IgG subclasses are less effective than their sdAb . Brain interstitial fluid (ISF) was collected from JNPL3 mice aged 7-11 months infused with 1D9 or 2B8 sdAb (n = 6 per group), whole IgG1 or IgG2A (n = 6 per group), or artificial CSF (aCSF) vehicle (n = 11). Baseline samples were collected during the animals’ inactive period, and then for a further 24 hours following antibody administration. The shaded boxes in panels A and C represent the typical dark (awake) cycle for the mice. Even though the animals were maintained in constant light, they display the typical diurnal pattern of tau release during the first 24 h. a. In mice receiving 1D9, there were significant antibody, time, and interaction effects (p < 0.0001, = 0.0088, < 0.0001 respectively, two-way ANOVA). 1D9 sdAb treated animals had lower ISF tau levels relative to untreated controls at 7-10 h post-infusion (p = 0.027 and 0.0055 for 7-8 h and 9-10 h). 1D9 IgG1 infused animals were lower than untreated mice from 3-18 h (p values range from 0.033 through < 0.0001), and IgG2A treated mice from 0-18 h (p values range from 0.045 through < 0.0001). 1D9 IgG1 samples were lower than their sdAb at 5-6 h and 9-10 h (p = 0.035 and 0.025). b. When post-infusion samples were pooled, 1D9 sdAb, IgG1 and IgG2A samples were all lower than untreated control samples (p < 0.0001 for all, overall p < 0.0001, one-way ANOVA). IgG1 and IgG2A were also more effective at preventing the activity related tau increase compared to the sdAb (p < 0.0001 for both). c. In mice receiving 2B8, there were significant treatment, time, and interaction effects (p < 0.0001, = 0.0033, < 0.0001, two-way ANOVA). 2B8 sdAb treated mice had lower ISF tau compared to untreated animals from 0-22 h (p values range from 0.011 through < 0.0001). 2B8 IgG1 treated mice had lower tau levels at 3-14 h (p values range from 0.035 through < 0.0001), and 2B8 IgG2A treated mice only at 5-8 h (p = 0.029 and 0.0001 at 5-6 h and 7-8 h). 2B8 IgG1 values were also higher than its sdAb at 11-12 h and 15-20 h (p values range from 0.048 through 0.0055), and 2B8 IgG2A values were higher at 9-20 h (p values range from 0.035 through < 0.0001). d. All pooled 2B8 sdAb samples had lower ISF tau than untreated controls (p < 0.0001, < 0.0001, = 0.0003, overall p < 0.0001, one-way ANOVA). Post infusion samples from mice treated with 2B8 IgG1 and IgG2A had higher tau levels compared to their sdAb (p = 0.004 and < 0.0001) Points and bars represent average ± SEM. #### p < 0.0001, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p < 0.0001. Panels a and c: * sdAb relative to untreated, * IgG1 relative to untreated, * IgG2A relative to untreated controls. Panels b and d: # significant compared to untreated control, * significant compared to sdAb.

Article Snippet: Axon Neuroscience generated IgG1 and IgG4 variants of AX004, a humanized version of truncated tau antibody DC8E8, and showed that the IgG1 was more effective than the IgG4 in promoting tau uptake in primary human microglial cultures.

Techniques: In Vivo, Control, Activity Assay

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control