Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis

In vitro selections of mammaglobin A and mammaglobin B aptamers for the recognition of circulating breast tumor cells

Mammaglobin B (MGB2) and mammaglobin A (MGB1) are proteins expressed in metastatic breast cancers. The early detection of circulating tumor cells (CTCs) in breast cancer patients is crucial to decrease mortality rate. Herein, novel aptamers were successfully selected and characterized agai

N-acetylcysteine manipulation fails to elicit an increase in glutathione in a teleost model

Levels of oxidative stress can be affected by a range of compounds including toxins and pharmaceuticals. Antioxidants are important protective compounds which counteract the damaging effects of oxidative stress. Glutathione (GSH) is one of the main antioxidants for many organisms and can be synthesized from administered N-acetylcysteine (NAC). NAC has therefore often been used in a wide range of taxa to manipulate levels of GSH. Our objective was to validate this approach in a wild temperate teleost fish model, the brown trout (Salmo trutta). We used intracoelomic injections of NAC in saline and vegetable shortening, at two different concentrations (100 and 400 mg/kg), with the appropriate controls and shams, under controlled laboratory settings. We found tha

Oxidative stress and partial migration in brown trout (Salmo trutta)

During migration, animals are typically limited by their endogenous energetic resources that must be allocated to the physiological costs associated with locomotion, as well as avoiding and (or) compensating for oxidative stress. To date, there have been few attempts to understand the role of oxidative status in migration biology, particularly in fish. Semi-anadromous brown trout (Salmo trutta L., 1758) exhibit partial migration, where some individuals smoltify and migrate to sea, and others become stream residents, providing us with an excellent model to investigate the link between oxidative stress and migration. Using the brown trout, we obtained blood samples from juveniles from a coastal stream in Denmark in the fall prior to peak seaward migration that occurs in the spring, and assayed for antioxidant capacity (oxygen radical absorbance capacity) and oxidative stress levels (ratio of oxidized to reduced glutathione). We found that individuals that migrated had higher antioxidant capacity than residents and that future migration date was negatively correlated with both antioxidant capacity and body length in the fall. This study provides the first evidence that oxidative status is associated with migration strategy and timing, months in advance of the actual migration, and provides insight into the role of oxidative status in animal migration

Morphological, physiological and dietary covariation in migratory and resident adult brown trout (Salmo trutta)

The causes and consequences of trait relationships within and among the categories of physiology, morphology, and life-history remain poorly studied. Few studies cross the boundaries of these categories, and recent reviews have pointed out not only the dearth of evidence for among-category correlations but that trait relationships may change depending on the ecological conditions a population faces. We examined changes in mean values and correlations between traits in a partially migrant population of brown trout when migrant sea-run and resident stream forms were breeding sympatrically. Within each sex and life-history strategy group, we used carbon and nitrogen stable isotopes to assess trophic level and habitat use; assessed morphology which reflects swimming and foraging ability; measured circulating cortisol as it is released in response to stressors and is involved in the transition from salt to freshwater; and determined oxidative status by measuring oxidative stress and antioxidants. We found that sea-run trout were larger and had higher values of stable isotopes, cortisol and oxidative stress compared to residents. Most groups showed some correlations between morphology and diet, indicating individual resource specialization was occurring, and we found consistent correlations between morphology and cortisol. Additionally, relationships differed between the sexes (cortisol and oxidative status were related in females but not males) and between life-history strategies (habitat use was related to oxidative status in male sea-run trout but not in either sex of residents). The differing patterns of covariation between the two life-history strategies and between the sexes suggest that the relationships among phenotypic traits are subjected to different selection pressures, illustrating the importance of integrating multiple phenotypic measures across different trait categories and contrasting life-history strategies

Rapid Detection of Circulating Breast Cancer Cells Using a Multiresonant Optical Fiber Aptasensor with Plasmonic Amplification

The detection of circulating tumor cells (CTCs), which are responsible for metastasis in several forms of cancer, represents an important goal in oncological diagnosis and treatment. These cells remain extremely challenging to detect, despite numerous previous studies, due to their low concentration (1-10 cells/mL of blood). In this work, an all-fiber plasmonic aptasensor featuring multiple narrowband resonances in the near-infrared wavelength range was developed to detect metastatic breast cancer cells. To this aim, specific aptamers against mammaglobin-A were selected and immobilized as receptors on the sensor surface. In vitro assays confirm that the label-free and real-time detection of cancer cells [limit of detection (LOD) of 49 cells/mL] occurs within 5 min, while the additional use of functionalized gold nanoparticles allows a 2-fold amplification of the biosensor response. Differential measurements on selected optical resonances were used to process the sensor response, and results were confirmed by microscopy. The detection of only 10 cancer cells/mL was achieved with relevant specificity against control cells and with quick response time