Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge.

Antibodies are one of the most important reagents used in biomedical and fundamental research, used to identify, and quantify proteins, contribute to knowledge of disease mechanisms, and validate drug targets. Yet many antibodies used in research do not recognize their intended target, or recognize additional molecules, compromising the integrity of research findings and leading to waste of resources, lack of reproducibility, failure of research projects, and delays in drug development. Researchers frequently use antibodies without confirming that they perform as intended in their application of interest. Here we argue that the determinants of end-user antibody choice and use are critical, and under-addressed, behavioral drivers of this problem. This interacts with the batch-to-batch variability of these biological reagents, and the paucity of available characterization data for most antibodies, making it more difficult for researchers to choose high quality reagents and perform necessary validation experiments. The open-science company YCharOS works with major antibody manufacturers and knockout cell line producers to characterize antibodies, identifying high-performing renewable antibodies for many targets in neuroscience. This shows the progress that can be made by stakeholders working together. However, their work so far applies to only a tiny fraction of available antibodies. Where characterization data exists, end-users need help to find and use it appropriately. While progress has been made in the context of technical solutions and antibody characterization, we argue that initiatives to make best practice behaviors by researchers more feasible, easy, and rewarding are needed. Global cooperation and coordination between multiple partners and stakeholders will be crucial to address the technical, policy, behavioral, and open data sharing challenges. We offer potential solutions by describing our Only Good Antibodies initiative, a community of researchers and partner organizations working toward the necessary change. We conclude with an open invitation for stakeholders, including researchers, to join our cause.

Effects of a single bout of strenuous exercise on platelet activation in female ApoE/LDLR-/- mice.

Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI2) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR-/- mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF1α, nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR-/- mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR-/- but not in WT mice, strenuous exercise partially inhibited TXB2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR-/- mice; however, only 7-month-old ApoE/LDLR-/- mice showed lower TXB2 production after exercise. In female 4-6-month-old ApoE/LDLR-/- but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO2-) and nitrate (NO3-) were decreased in ApoE/LDLR-/- as compared to that in age-matched WT mice. In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR-/- as compared to WT mice. However, platelet activation in ApoE/LDLR-/- mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI2-dependent anti-platelet mechanisms in ApoE/LDLR-/- mice.

Skeletal Muscle Response to Endurance Training in IL-6-/- Mice.

We examined effects of moderate-intensity endurance training on muscle COX/CS activities and V'O2max in control WT and IL-6(-/-) mice. Animals were exercised for 10 weeks on treadmill for 1 h, 5 days a week at velocity of 6 m·min(-1) which was increased by 0.5 m·min(-1) every 2 weeks up to 8 m·min(-1) . Training triggered an increase of enzyme activities in soleus muscle of WT mice (COX: 480.3±8.9 U·g(-1) in sedentary group vs. 773.3±62.6 U·g(-1) in trained group, P<0.05 and CS: 374.0±6.0 U·g(-1) in sedentary group vs. 534.2±20.5 U·g(-1) in trained group, P<0.01, respectively) whereas no changes were observed in soleus of IL6(-/-) mice. Moreover, in mixed gastrocnemius muscle of trained IL-6(-/-) mice enzyme activities tended to be lower (COX: 410.7±48.4 U·g(-1) for sedentary vs. 277.0±36.5 U·g(-1) for trained group and CS: 343.8±24.6 U·g(-1) for sedentary vs. 251.7±27.1 U·g(-1) for trained group). No changes in V'O2max were observed in WT and IL-6(-/-) mice after training. Concluding, moderate-velocity endurance training-induced increase in COX and CS activities in muscles of WT mice only which suggests that IL-6 regulates training-induced skeletal muscle responses to exercise.

Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions.

A major barrier to the use of nitrogen-fixing inoculum strains for the enhancement of legume productivity is the inability of commercially available strains to compete with indigenous rhizobia for nodule formation. Despite extensive research on nodulation competitiveness, there are no examples of field efficacy studies of strains that have been genetically improved for nodulation competitiveness. We have shown previously that production of the peptide antibiotic trifolitoxin (TFX) by Rhizobium etli results in significantly increased nodule occupancy values in nonsterile soil in growth chamber experiments (E. A. Robleto, A. J. Scupham, and E. W. Triplett, Mol. Plant-Microbe Interact. 10:228-233, 1997). To determine whether TFX production by Rhizobium etli increases nodulation competitiveness in field-grown plants, seeds of Phaseolus vulgaris were inoculated with mixtures of Rhizobium etli strains at different ratios. The three nearly isogenic inoculum strains used included TFX-producing and non-TFX-producing strains, as well as a TFX-sensitive reference strain. Data was obtained over 2 years for nodule occupancy and over 3 years for assessment of the effect of the TFX production phenotype on grain yield. In comparable mixtures in which the test strain accounted for between 5 and 50% of the inoculum, the TFX-producing strain exhibited at least 20% greater nodule occupancy than the non-TFX-producing strain in both years. The TFX production phenotype had no effect on grain yield over 3 years; the average yields reached 2,400 kg/ha. These results show that addition of the TFX production phenotype significantly increases nodule occupancy under field conditions without adverse effects on grain yield. As we used common inoculation methods in this work, there are no practical barriers to the commercial adoption of the TFX system for agriculture.