Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and co-localization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

Environmental exposures associated with honey bee health.

Bee health is declining on a global scale, yet the exact causes and their interactions responsible for the decline remain unknown. To more objectively study bee health, recently biomarkers have been proposed as an essential tool, because they can be rapidly quantified and standardized, serving as a comparable measure across bee species and varying environments. Here, we used a systems biology approach to draw associations between endogenous and exogenous chemical profiles, with pesticide exposure, or the abundance of the 21 most common honey bee diseases. From the analysis we identified chemical biomarkers for both pesticide exposure and bee diseases along with the mechanistic biological pathways that may influence disease onset and progression. We found a total of 2352 chemical features, from 30 different hives, sampled from seven different locations. Of these, a total of 1088 significant associations were found that could serve as chemical biomarker profiles for predicting both pesticide exposure and the presence of diseases in a bee colony. In almost all cases we found novel external environmental exposures within the top seven associations with bee diseases and pesticide exposures, with the majority having previously unknown connections to bee health. We highlight the exposure-outcome paradigm and its ability to identify previously uncategorized interactions from different environmental exposures associated with bee diseases, pesticides, mechanisms, and potential synergistic interactions of these that are responsible for honey bee health decline.

COVID-19 diagnosis -A review of current methods.

A fast and accurate self-testing tool for COVID-19 diagnosis has become a prerequisite to comprehend the exact number of cases worldwide and to take medical and governmental actions accordingly. SARS-CoV-2 (formerly, 2019-nCoV) infection was first reported in Wuhan (China) in December 2019, and then it has rapidly spread around the world, causing ~14 million active cases with ~582,000 deaths as of July 2020. The diagnosis tools available so far have been based on a) viral gene detection, b) human antibody detection, and c) viral antigen detection, among which the viral gene detection by RT-PCR has been found as the most reliable technique. In this report, the current SARS-CoV-2 detection kits, exclusively the ones that were issued an "Emergency Use Authorization" from the U.S. Food and Drug Administration, were discussed. The key structural components of the virus were presented to provide the audience with an understanding of the scientific principles behind the testing tools. The methods that are still in the early research state were also reviewed in a subsection based on the reports available so far.

Age-Dependent Honey Bee Appetite Regulation Is Mediated by Trehalose and Octopamine Baseline Levels.

There are multiple feedback mechanisms involved in appetite regulation, which is an integral part of maintaining energetic homeostasis. Older forager honey bees, in comparison to newly emerged bees and nurse bees, are known to have highly fluctuating hemolymph trehalose levels, higher appetite changes due to starvation, and higher octopamine levels in the brain. What remains unknown is if the hemolymph trehalose and octopamine levels interact with one another and how this varies as the bee ages. We manipulated trehalose and octopamine levels across age using physiological injections and found that nurse and forager bees increase their appetite levels due to increased octopamine levels in the brain. This is further enhanced by lower trehalose levels in the hemolymph. Moreover, nurse bees with high octopamine levels in the brain and low trehalose levels had the same appetite levels as untreated forager bees. Our findings suggest that the naturally higher levels of octopamine as the bee ages may result in higher sensitivity to fluctuating trehalose levels in the hemolymph that results in a more direct way of assessing the energetic state of the individual. Consequently, forager bees have a mechanism for more precise regulation of appetite in comparison to newly emerged and nurse bees.