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Calibrated brightfield-based imaging for measuring intracellular protein concentration03/21/2017Intracellular protein concentration is an essential cell characteristic which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/l). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (m3) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Furthermore, interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle. Our data on HeLa, MDCK and DU145 cells indicate that it remains relatively stable. |
Brain-induced muscle thermogenesis is accompanied by increased sarcolipin expression in skeletal muscle03/21/2017Obesity is a major health risk that can lead to numerous complications such as heart disease and type II diabetes. In order to find a way to counter the obesity pandemic and promote weight loss, thermogenic mechanisms were examined as a method to burn caloric energy as heat in skeletal muscle, a novel approach. In the brain, both the melanocortin system and the ventromedial hypothalamus play important roles in energy balance and thermogenesis. The activation of melanocortin receptors in this brain region induced heat dissipation in skeletal muscle. Within skeletal myocytes, sarcolipin modulates sarcoplasmic reticulum calcium ATPase (SERCA), uncoupling energy use from calcium cycling. Here, we investigated the expression of sarcolipin to determine its potential as a modulator of centrally induced thermogenesis within skeletal muscle. Quantitative PCR was used to compare sarcolipin expression in gastrocnemius muscle from rats that had been given brain injections of melanotan II, a melanocortin receptor agonist, to the sarcolipin expression in rat muscle that received vehicle control injections. There was significantly higher muscle sarcolipin mRNA expression in the rats that had received brain injections of melanotan II than those that did not. This implicates muscle sarcolipin as a driver of melanocortin-induced thermogenesis. Activating this thermogenic mechanism in skeletal muscle has exciting potential to work as an effective way to burn calories and help combat obesity. |
Altered circadian phenotype in cannabinoid receptor 1 knockout mice03/21/2017The suprachiasmatic nucleus of the hypothalamus (SCN) drives daily rhythms of physiology and behavior. Previous studies suggest a role for cannabinoids in regulating circadian rhythms, acting through the cannabinoid type 1 receptor (CB1). To further evaluate endocannabinoids in clock regulation, we screened CB1 knockout mice (CB1-/-) for circadian clock abnormalities. These mice show increased circadian disorganization, with some displaying increased wheel-running activity during the light phase of the light-dark cycle. In both constant dark and in constant light, CB1-/- mice show free-running periods that are significantly shorter than in wild-type controls. CB1-/- mice also show increased phase shifts in response to light pulses compared to control mice. Next, we evaluated the response of the mice to 6 hour phase advance and delays of the light-dark cycle (simulated jet lag). Reentrainment rates appeared to be faster in the CB1-/- mice, however, this was difficult to evaluate due to disruption of the rhythmic activity pattern. We then evaluated food anticipatory activity (FAA) during timed restricted feeding in mice under both a light-dark cycle and in constant light. Because CB1-/- mice show a very lean phenotype, we expected to find reduced FAA. Results suggest that CB1-/- show FAA that is equal to or greater than wild type mice. Finally, we assessed neuronal activation in the SCN in response to light pulses using expression of the gene Fos, but found no significant differences between CB1-/- and wild type mice. These data suggest that cannabinoids alter clock function through actions downstream of light input pathways. |
Agricultural Impacts on Aquatic Invertebrate Diversity and Abundance in Costa Rica03/21/2017Freshwater ecosystems are highly threatened by agricultural practices due to pesticide and herbicide runoff, which can have a strong effect on aquatic invertebrate communities due to them being sensitive to environmental changes and water quality. The influence of agriculture on aquatic ecosystems has been studied at length in temperate regions, but the consequences of this practice have not yet been explored in tropical regions. The purpose of this study is to compare the abundance and diversity of aquatic invertebrates between agricultural and non-agricultural (or naturally wooded) streams in Costa Rica to determine how agricultural practices influence aquatic invertebrate communities in the tropics. This experiment was conducted at the Alberto Manuel Brenes Biological Reserve in Costa Rica, which includes 7,800 Ha of unaltered forest, but the area surrounding the reserve has experienced an intense increase in agricultural practices. In August, 2016, samples were taken on 3 separate dates at 6 streams within the reserve and 3 within the agricultural land. Streams were scored using the Biological Monitoring Working Party Method developed in Costa Rica. Both indicator score (p=0.004) and indicator category (p=0.002) were significantly different between agricultural and non-agricultural streams. While the diversity of the non-agricultural streams was higher than the diversity of the agricultural streams (p=0.026), the abundance between treatments was not significant (p=0.321). While diversity was significantly different between treatments, abundance was not, indicating that it is not the amount of invertebrates but the type that indicates stream quality. |