Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.     

The Impact of Variants in Four Genes: MC4R,FTO, PPARG and PPARGC1A in Overweight and Obesity in a Large Sample of the Brazilian Population

Biochemical Genetics - Obesity and overweight are worldwide public health problems with an evident genetic predisposition that is still poorly understood. In addition, great variability has been...     

The acute systemic toxicity of thallium in rats produces oxidative stress: attenuation by metallothionein and Prussian blue

BioMetals - Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental...     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of I_Kir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.     

Late‐in‐life treadmill training rejuvenates autophagy,protein aggregate clearance,and function in mouse hearts

Protein quality control mechanisms decline during the process of cardiac aging. This enables the accumulation of protein aggregates and damaged organelles that contribute to age‐associated cardiac dysfunction. Macroautophagy is the process by which post‐mitotic cells such as cardiomyocytes clear defective proteins and organelles. We hypothesized that late‐in‐life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs. adult mice. As expected, 24‐month‐old male C57BL/6J mice (old) exhibited repressed autophagosome formation and protein aggregate accumulation in the heart, systolic and diastolic dysfunction, and reduced exercise capacity vs. 8‐month‐old (adult) mice (all p < 0.05). To investigate the influence of late‐in‐life exercise training, additional cohorts of 21‐month‐old mice did (old‐ETR) or did not (old‐SED) complete a 3‐month progressive resistance treadmill running program. Body composition, exercise capacity, and soleus muscle citrate synthase activity improved in old‐ETR vs. old‐SED mice at 24 months (all p < 0.05). Importantly, protein expression of autophagy markers indicate trafficking of the autophagosome to the lysosome increased, protein aggregate clearance improved, and overall function was enhanced (all p < 0.05) in hearts from old‐ETR vs. old‐SED mice. These data provide the first evidence that a physiological intervention initiated late‐in‐life improves autophagic flux, protein aggregate clearance, and contractile performance in mouse hearts.     

A field guide to cultivating computational biology

Evolving in sync with the computation revolution over the past 30 years, computational biology has emerged as a mature scientific field. While the field has made major contributions toward improving scientific knowledge and human health, individual computational biology practitioners at various institutions often languish in career development. As optimistic biologists passionate about the future of our field, we propose solutions for both eager and reluctant individual scientists, institutions, publishers, funding agencies, and educators to fully embrace computational biology. We believe that in order to pave the way for the next generation of discoveries, we need to improve recognition for computational biologists and better align pathways of career success with pathways of scientific progress. With 10 outlined steps, we call on all adjacent fields to move away from the traditional individual, single-discipline investigator research model and embrace multidisciplinary, data-driven, team science.

Do you want to attract computational biologists to your project or to your department? Despite the major contributions of computational biology, those attempting to bridge the interdisciplinary gap often languish in career advancement, publication, and grant review. Here, sixteen computational biologists around the globe present "A field guide to cultivating computational biology," focusing on solutions.

Biology in the digital era requires computation and collaboration. A modern research project may include multiple model systems, use multiple assay technologies, collect varying data types, and require complex computational strategies, which together make effective design and execution difficult or impossible for any individual scientist. While some labs, institutions, funding bodies, publishers, and other educators have already embraced a team science model in computational biology and thrived [1–7], others who have not yet fully adopted it risk severely lagging behind the cutting edge. We propose a general solution: “deep integration” between biology and the computational sciences. Many different collaborative models can yield deep integration, and different problems require different approaches (Fig 1).Open in a separate windowFig 1Supporting interdisciplinary team science will accelerate biological discoveries.Scientists who have little exposure to different fields build silos, in which they perform science without external input. To solve hard problems and to extend your impact, collaborate with diverse scientists, communicate effectively, recognize the importance of core facilities, and embrace research parasitism. In biologically focused parasitism, wet lab biologists use existing computational tools to solve problems; in computationally focused parasitism, primarily dry lab biologists analyze publicly available data. Both strategies maximize the use and societal benefit of scientific data.In this article, we define computational science extremely broadly to include all quantitative approaches such as computer science, statistics, machine learning, and mathematics. We also define biology broadly, including any scientific inquiry pertaining to life and its many complications. A harmonious deep integration between biology and computer science requires action—we outline 10 immediate calls to action in this article and aim our speech directly at individual scientists, institutions, funding agencies, and publishers in an attempt to shift perspectives and enable action toward accepting and embracing computational biology as a mature, necessary, and inevitable discipline (Box 1).Box 1. Ten calls to action for individual scientists, funding bodies, publishers, and institutions to cultivate computational biology. Many actions require increased funding support, while others require a perspective shift. For those actions that require funding, we believe convincing the community of need is the first step toward agencies and systems allocating sufficient support

1. Respect collaborators’ specific research interests and motivationsProblem: Researchers face conflicts when their goals do not align with collaborators. For example, projects with routine analyses provide little benefit for computational biologists.Solution: Explicit discussion about interests/expertise/goals at project onset.Opportunity: Clearly defined expectations identify gaps, provide commitment to mutual benefit.
2. Seek necessary input during project design and throughout the project life cycleProblem: Modern research projects require multiple experts spanning the project’s complexity.Solution: Engage complementary scientists with necessary expertise throughout the entire project life cycle.Opportunity: Better designed and controlled studies with higher likelihood for success.
3. Provide and preserve budgets for computational biologists’ workProblem: The perception that analysis is “free” leads to collaborator budget cuts.Solution: When budget cuts are necessary, ensure that they are spread evenly.Opportunity: More accurate, reproducible, and trustworthy computational analyses.
4. Downplay publication author order as an evaluation metric for computational biologistsProblem: Computational biologist roles on publications are poorly understood and undervalued.Solution: Journals provide more equitable opportunities, funding bodies and institutions improve understanding of the importance of team science, scientists educate each other.Opportunity: Engage more computational biologist collaborators, provide opportunities for more high-impact work.
5. Value software as an academic productProblem: Software is relatively undervalued and can end up poorly maintained and supported, wasting the time put into its creation.Solution: Scientists cite software, and funding bodies provide more software funding opportunities.Opportunity: More high-quality maintainable biology software will save time, reduce reimplementation, and increase analysis reproducibility.
6. Establish academic structures and review panels that specifically reward team scienceProblem: Current mechanisms do not consistently reward multidisciplinary work.Solution: Separate evaluation structures to better align peer review to reward indicators of team science.Opportunity: More collaboration to attack complex multidisciplinary problems.
7. Develop and reward cross-disciplinary training and mentoringProblem: Academic labs and institutions are often insufficiently equipped to provide training to tackle the next generation of biological problems, which require computational skills.Solution: Create better training programs aligned to necessary on-the-job skills with an emphasis on communication, encourage wet/dry co-mentorship, and engage younger students to pursue computational biology.Opportunity: Interdisciplinary students uncover important insights in their own data.
8. Support computing and experimental infrastructure to empower computational biologistsProblem: Individual computational labs often fund suboptimal cluster computing systems and lack access to data generation facilities.Solution: Institutions can support centralized compute and engage core facilities to provide data services.Opportunity: Time and cost savings for often overlooked administrative tasks.
9. Provide incentives and mechanisms to share open data to empower discovery through reanalysisProblem: Data are often siloed and have untapped potential.Solution: Provide institutional data storage with standardized identifiers and provide separate funding mechanisms and publishing venues for data reuse.Opportunity: Foster new breed of researchers, “research parasites,” who will integrate multimodal data and enhance mechanistic insights.
10. Consider infrastructural, ethical, and cultural barriers to clinical data accessProblem: Identifiable health data, which include sensitive information that must be kept hidden, are distributed and disorganized, and thus underutilized.Solution: Leadership must enforce policies to share deidentifiable data with interoperable metadata identifiers.Opportunity: Derive new insights from multimodal data integration and build datasets with increased power to make biological discoveries.