The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.
Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.
Most climate change studies that address potential impacts and potential adaptation strategies are largely based on modelling technologies. While models are useful for visualizing potential future outcomes and evaluating options for potential adaptation, they do not adequately represent and integrate adaptive human agency. Richards’ concept of ‘agriculture as performance’ is useful in counterbalancing the modelling approach to adaptation because it highlights how adaptive processes and technologies, whether short term or long term, are more than simple technical responses to biophysical conditions. Instead, adaptive processes are social phenomena whose significance and effects expand well beyond changing climate conditions. This examination of agriculture as performance in the context of climate adaptation draws on two different examples. The first example explores how technical aspects of climate adaptation in Mali are situated within the enactment of ethnic identities and political struggles between farmers and herders. The second example shows how farmers in southeastern United States approach climate variability and climate forecasts as risk management tools. There are substantial differences between approaching adaptation as a dynamic process that is socially embedded and approaching adaptation as a set of modelled responses to anticipated future conditions. It is unlikely that either is adequate to meet the challenges posed by the uncertainties associated with climate change. However, building a synergistic relationship between the two promises to be as difficult as it is necessary.
In the agricultural setting, core global food safety elements, such as hand hygiene and worker furlough, should reduce the risk of norovirus contamination on fresh produce. However, the effect of these practices has not been characterized. Using a quantitative microbial risk model, we evaluated the individual and combined effect of farm-based hand hygiene and worker furlough practices on the maximum risk of norovirus infection from three produce commodities (open leaf lettuce, vine tomatoes, and raspberries). Specifically, we tested two scenarios where a harvester's and packer's norovirus infection status was: 1) assumed positive; or 2) assigned based on community norovirus prevalence estimates. In the first scenario with a norovirus-positive harvester and packer, none of the individual interventions modeled reduced produce contamination to below the norovirus infectious dose. However, combined interventions, particularly high handwashing compliance (100%) and efficacy (6 log10 virus removal achieved using soap and water for 30 s), reduced produce contamination to <1–82 residual virus. Translating produce contamination to maximum consumer infection risk, 100% handwashing with a 5 log10 virus removal was necessary to achieve an infection risk below the threshold of 0.032 infections per consumption event. When community-based norovirus prevalence estimates were applied to the harvester and packer, the single interventions of 100% handwashing with 3 log10 virus removal (average 0.02 infection risk per consumption event) or furlough of the packer (average 0.03 infection risk per consumption event) reduced maximum infection risk to below the 0.032 threshold for all commodities. Bundled interventions (worker furlough, 100% glove compliance, and 100% handwashing with 1-log10 virus reduction) resulted in a maximum risk of 0.02 per consumption event across all commodities. These results advance the evidence-base for global produce safety standards as effective norovirus contamination and risk mitigation strategies.
Imbalances in bone formation and resorption cause osteoporosis. Mounting evidence supports that brain-derived neurotrophic factor (BDNF) implicates in this process. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived small molecular TrkB agonist, mimics the functions of BDNF. We show that both BDNF and 7,8-DHF promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. These effects might be attributed to the activation of the Wnt/β-catenin signaling pathway as the expression of cyclin D1, phosphorylated-glycogen synthase kinase-3β (p-GSK3β), β-catenin, Runx2, Osterix, and osteoprotegerin (OPG) was all significantly up-regulated. Knockdown of β-catenin restrained the up-regulation of Runx2 and Osterix stimulated by 7,8-DHF. In particular, blocking TrkB by its specific inhibitor K252a suppressed 7,8-DHF-induced osteoblastic proliferation, differentiation, and expression of osteoblastogenic genes. Moreover, BDNF and 7,8-DHF repressed osteoclastic differentiation of RAW264.7 cells. The transcription factor c-fos and osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), matrix metalloprotein-9 (MMP-9), Adamts5 were inhibited by 7,8-DHF. More importantly, 7,8-DHF attenuated bone loss, improved trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in an ovariectomy (OVX) rat model. The current work highlights the dual regulatory effects that 7,8-DHF exerts on bone remodeling.